Methods and compositions involving fibrillizing polypeptides for nanofibers

ABSTRACT

Embodiments of the invention are directed to fibrillar adjuvants. For example, epitopes assembled by a synthetic peptide domain into nanofibers comprising a β-fibrillization peptide may elicit high antibody titers in the absence of any adjuvant. In certain embodiments, multiple different antigens may be integrated into polypeptide nanofibers, providing biomaterials with modular and precise composition of bioactive proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 14/209,757 filed Mar. 13, 2014, which claimspriority to U.S. Provisional Application Ser. No. 61/782,193 filed onMar. 14, 2013. The entire contents of each of the above-referenceddisclosures are specifically incorporated herein by reference withoutdisclaimer.

This invention was made with government support under R01 EB009701 and1R21AI094444 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of this invention are directed generally to biology,medicine, and immunology. Certain aspects are directed to immunogenicfibrils and their use in inducing an immune response.

2. Description of Related Art

Polypeptides that non-covalently assemble into supramolecularstructures, such as nanofibers and nanoparticles, are receivingincreased interest as biomaterials for diverse applications, includingenzyme catalysis (Wheeldon 2009; Baxa 2002; Patterson 2012; Guglielmi2009) biosensors (Leng 2010; Men 2009), electronics (Baldwin 2006;Wheeldon 2008), tissue engineering (Wang 2011; Horii 2007), drugs anddrug delivery (Webber 2012; Matson 2011; Sinthuvanich 2012), andimmunotherapy (Rudra 2010; Hudalla 2013; Black 2012; Wahome 2012). Inpart, this widespread applicability arises from the ability toincorporate a self-assembling domain and a bioactive ligand, such as apeptide, protein, or nucleic acid, into a single molecule viarecombinant genetic fusion or chemical synthesis approaches, withoutperturbing the assembly or bioactive properties of the respectivedomains (Cardinale 2012; Lim 2009; Woolfson 2010; Guler 2005). Inaddition, mixtures of self-assembling polypeptides with or withoutappended bioactive ligands co-assemble into multi-componentbiomaterials, in which molecular composition is governed by the molarratio of polypeptides present during assembly (Collier 2008; Collier2010; Matson 2012; Minten 2009; Minten 2011). Importantly, this preciseand reproducible compositional control enables use of statisticalmethods to identify ligand formulations that elicit optimal functionalresponses (Jung 2011), which can be challenging to achieve withco-polymer blends that are subject to compositional drift. However,supramolecular assemblies bearing multiple different folded proteinligands at precise concentrations have not yet been realized, despiteexisting approaches to create assemblies having tunable concentration ofa single protein ligand (Hudalla 2013; Minten 2009; Sangiambut 2013), orbearing two different biologically active proteins (Leng 2010; Men 2009;Minten 2011). A general approach that enables modular and tunablecontrol over integrated protein ligand composition would provideenormous nanofiber design flexibility, ultimately leading to newbiomaterials with unique biological or chemical properties for variousdownstream applications.

SUMMARY OF THE INVENTION

Certain embodiments are directed to a nanofiber composition comprising aβ-sheet nanofiber structure. The structure may have a length of atleast, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200 nm,0.25, 0.5, 1, 10, 50 to 10, 25, 50, 100 μm, including all values andranges there between. In certain aspects, the composition has amolecular weight of at least about 1,000, 5,000, 10,000, 100,000 Da toabout 1×10⁶, 1×10⁷, 7×10⁸ Da, including all values and ranges therebetween.

In certain aspects, the β-sheet nanofiber comprises a plurality of apeptide A and a peptide B. The peptide A may be non-β-sheet peptide tagssuch as a β-sheet fibrillizing tail (a “βTail) exemplified herein. Thenon-β-sheet peptide may refer to a peptide that forms a structure otherthan a β-sheet structure when expressed or isolated. The non-β-sheetpeptide may be an α-helical peptide or random coil peptide whenexpressed or isolated. However, the non-β-sheet peptide tags may form aβ-sheet structure in the presence of a β-sheet peptide.

In alternative embodiments, the peptide A may be β-sheet peptides (e.g.,Q11 peptide) and may be attached to a compound, such as in the form of afusion protein. A plurality of a peptide A, each is a β-sheet peptideattached to a compound, may form a nanofiber with a plurality of peptideB, which are β-sheet peptides on their own.

In further aspects, the β-sheet nanofiber comprises a peptide B such asa β-sheet peptide. The β-sheet peptide may refer to a peptide that formsa β-sheet structure. The β-sheet peptides may integrate the non-β-sheetpeptide tags into a nanofiber structure.

One or more non-β-sheet peptides may be attached as a tag to one or morecompounds. It is contemplated that a single tag (such as a single β-tailpeptide molecule) may be attached to 1, 2, 3 or more compounds, whichmay be the same or different with respect to one another. Whilecompositions comprise a plurality of tags, in some embodiments there aresame or different tags that are used. Different tags may be attached tothe same or to different compounds. Same tags may be attached to thesame or to different compounds, for example, each of a subset of theβ-tail molecules is attached to a dGFP compound, and each of a subset ofthe β-tail molecules is attached to a RFP compound, and each of a subsetof the β-tail molecules is attached to a dGFP compound. The molar ratiobetween the non-β-sheet peptide tag and any compound may be 1:1. 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or lower such as 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or any intermediate ranges. In certainaspects, the composition or structure may be heterogeneous by comprisingat least two, three, four, five, six, seven, nine, ten, or more (or anyrange derivable therein) different compounds. Different compounds may beattached to the same or to different tags. Same compounds may beattached to the same or to different compounds. Moreover, there may be1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (or any range derivable therein)different tags.

In some embodiments, the non-β-sheet peptides may comprise one or moreα-helical motifs such as coiled-coil motifs. The α-helical motifs mayhave a sequence of a b c d e f g. The sequence motif may be repeated forabout two to seven times. The sequence motifs may be repeated with thesame generic structure as described below and same or different specificsequences.

In one, two, three, four, five, six, seven or more or all of thesequence motifs, the position a and d may be non-polar amino acids,hydrophobic amino acids, or non-charged amino acids. For example, aand/or d is Ala (A), Leu (L), Ile (I), Val (V), or a conservativederivative thereof. In particular embodiments, a and/or d is Leu (L) inone, two, three, four, five, six, seven or more or all of the sequencemotifs.

In one, two, three, four, five, six, seven or more or all of thesequence motifs, the positions e and g may be charged amino acids, suchas Lys (K), Arg (R), His (H), Asp (D), Glu (E) or a conservativederivative thereof. For example, e and g may form or not form attractiveelectrostatic interactions in one, two, three, four, five, six, seven ormore or all of the sequence motifs.

In particular embodiments, one or more of b, c, and, f is a hydrophobicamino acid that favors β-sheet formation or increase β-sheet formationpropensity in one, two, three, four, five, six, seven or more or all ofthe sequence motifs. For example, one or more of b, c, and, f in one ormore of the α-helical motifs is Val (V), Tyr (Y), Phe (F), Trp (W), Ile(I), or Thr (T) in one, two, three, four, five, six, seven or more orall of the sequence motifs. More particularly, b, c, and f arebeta-sheet forming residues such as Val (V) in one, two, three, four,five, six, seven or more or all of the sequence motifs.

In further aspects, the non-β-sheet peptide comprises an amino acidsequence having at least 50, 60, 70, 75, 80, 85, 90, 95, 99, 100%identity (or any intermediate ranges) with the sequence ofLVVLHSELHKLKSEL (SEQ ID NO. 1), LVVLHSHLEKLKSEL (SEQ ID NO. 2),LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO. 3), LKVELEKLKSELVVLHSHLEKLKSEL(SEQ ID NO. 4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO. 5). In certainaspects, the non-β-sheet peptide is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15to 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500amino acids in length, including all values and ranges there between.

In certain aspects, one or more of the alpha helical motifs of thenon-β-sheet peptide may further comprise at least one, two, three, four,five, six, seven, eight, nine, ten metal binding amino acids or anyrange derivable therein. Each of the two metal binding amino acids inone or more of the alpha helical motifs or the non-β-sheet peptide maybe spaced by at least one, two, three, four, five, six, seven, eight,nine, ten amino acids or any range derivable therein. The spacing aminoacids may be any amino acids, hydrophobic, hydrophilic, charged,metal-binding, or not. The non-β-sheet peptide may also not need anymetal binding amino acids, such as LKVELKELKKELVVLKSELKELKKEL (SEQ IDNO. 5).

In particular aspects, one or more of the alpha helical motifs of thenon-β-sheet peptide further comprise at least two metal binding aminoacids spaced by one amino acid. For example, the non-β-sheet peptide maycomprise an amino acid sequence having at least or about 50, 60, 70, 75,80, 85, 90, 95, 99, or 100% identity (or any range derivable therein)with the sequence of LVVLHSHLEKLKSEL (SEQ ID NO. 2) orLKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO. 4).

In further aspects, one or more of the alpha helical motifs furthercomprise at least two metal binding amino acids spaced by three aminoacids. For example, the non-β-sheet peptide may comprise an amino acidsequence having at least or about 50, 60, 70, 75, 80, 85, 90, 95, 99, or100% identity (or any range derivable therein) with the sequence ofLVVLHSELHKLKSEL (SEQ ID NO. 1) or LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO.3).

In further aspects, there may be provided a composition comprising ananofiber comprising peptide A and peptide B. In further aspects, thepeptide A may be attached to any compound at a molar ratio of 1:1. 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or lower such as 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or any intermediate ranges. The nanofibermay comprise the same compound or two, three, four, five, six or moredifferent compounds.

The peptide A may comprise an amino acid sequence having at least orabout 50, 60, 70, 75, 80, 85, 90, 95, 99, or 100% identity (or any rangederivable therein) with the sequence of LVVLHSELHKLKSEL (SEQ ID NO. 1),LVVLHSHLEKLKSEL (SEQ ID NO. 2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO.3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO. 4), orLKVELKELKKELVVLKSELKELKKEL (SEQ ID NO. 5).

The peptide B may comprise an amino acid sequence having at least orabout 50, 60, 70, 80, 85, 90, 95, 99, or 100% identity (or any rangederivable therein) with the sequence of QQKFQFQFEQQ (SEQ ID NO. 6);QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO.9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11);FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13);QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE(SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ IDNO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO.19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21);SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (WhereX=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P)(SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO.25); LLLLKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO.27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV(SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO.32); or YTIAALLSPY (SEQ ID NO. 33).

For example, the peptide A may be at least, at most or about 15, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100 amino acids in length, including allvalues and ranges there between. In further aspects, the peptide A maybe at least, at most or about 7, 8, 9, 10, 11, 12, 13, 14, 15 to 15, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100 amino acids in length, includingall values and ranges there between.

In certain aspects, one or more of the compounds attached to the non-βsheet peptide tags (or peptide A) may be peptides, polypeptides, nucleicacids, small molecules, antigens, ligands, enzymes, reporters, drugs,matrices, cells, viruses, bacteria, lipids, carbohydrates, or acombination thereof.

For example, one or more of the compounds may be a peptide, same ordifferent. In further aspects, at least one, two, three, four, more orall of the non-β-sheet peptide tags attached to a compound is a fusionprotein. The non-β-sheet peptide tag (or peptide A) may be attached tothe N and/C terminus of a peptide compound. In particular aspects, thenon-β-sheet peptide tag (or peptide A) is attached to a peptide having 2to 10,000 amino acids in length, more particularly 5, 10, 15, 20 to 15,20, 30, 40, 50, 100, 200, 300, 400, 500, 1000 amino acids in length,including all values and ranges there between.

Non-limiting examples of a peptide compound attached to a non-β-sheetpeptide tag (or peptide A) include an enzyme, fluorescent protein, cellbinding domain, cell adhesion domain, extracellular matrix domain,reporter protein, cytokine, antigen, signaling domain, immunomodulatingprotein, cross-linking protein, hormone, hapten, or a combinationthereof. In a particular example, extracellular matrix proteins orextracellular matrix protein domains may be used as the peptide compoundor in the composition.

As used herein, the term “extracellular matrix”, abbreviated “ECM”,refers to the complex structural material that is produced by cells inmammalian tissues, particularly cells of connective tissue, for instancesuch cells as fibroblasts, osteoblasts, chondrocytes, epithelial cells,smooth muscle cells, adipocytes, and mesenchymal cells, and whichmaterial in vivo surrounds and supports those cells. Typically, the ECMis composed of fibres embedded in what is commonly referred to as‘ground substance’. ECM proteins include proteins in the fibers asstructural proteins, such as collagen and/or elastin. Particularlysuitable collagens are fibril-forming collagens. Type I collagen, typeII collagen, type III collagen, type IV collagen or type X collagen areparticularly preferred. A particular example is type I collagen.

ECM proteins also include proteins in the ‘ground substance’ of ECM,such as fibrillin, fibronectin, and/or laminin. Additional ECM proteinsmay include: glycoproteins such as laminin, entactin, tenascin,fibrillin, or fibronectin, for improving structural integrity of thenetwork and for the attachment of cells to the ECM; osteocalcin (GIaprotein), as a protein that binds calcium during mineralization;osteonectin, which serves a bridging function between collagen andmineral component; and sialoproteins, such as bone sialoprotein (BSP),osteopontin (OPN), dentin matrix protein-1 (DMP1), dentinsialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein(MEPE).

When used in certain aspects, the term “extracellular matrix” or“extracellular matrix protein or protein domain” refers both to thematerial in vivo, as well as to the material in isolated form, separatedfrom the cells that produced it. The ECM in certain aspects can be anatural or artificial material (e.g., a proteinaceous or peptidehydrogel).

In particular aspects, the compound attached to one or more of thenon-β-sheet peptide tags (or peptide A) is an antigen. Antigens can bemicrobial antigens, such as viral, fungal, or bacterial; or therapeuticantigens such as antigens associated with cancerous cells or growths,including tumor antigens, or autoimmune disorders. In certain aspects,the antigens are peptides, lipids, carbohydrates, or other immunogenicmolecules. The antigens can be T-cell and/or B-cell epitopes.

As used herein, the term “antigen” is a molecule capable of inducing animmune response or of being bound by an antibody or T-cell receptor. Anantigen is additionally capable of inducing a humoral immune responseand/or cellular immune response leading to the production of B- and/orT-lymphocytes. The structural aspect of an antigen that gives rise to abiological response is referred to herein as an “antigenic determinant.”B-lymphocytes respond to foreign antigenic determinants via antibodyproduction, whereas T-lymphocytes are the mediator of cellular immunity.Thus, antigenic determinants or epitopes are those parts of an antigenthat are recognized by antibodies, or in the context of an MHC, byT-cell receptors. An antigenic determinant need not be a contiguoussequence or segment of protein and may include various sequences thatare not immediately adjacent to one another. In particular aspects, theantigen may be an antigenic determinant or epitope.

In further aspects, the non-β-sheet peptide tags (or peptide A) andβ-sheet peptides (or peptide B) may have a molar ratio of about 1:1,1:2, 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:5000, 1:10,00050,000 to about 1:100,000 in the composition or the nanofiber structure,including all values and ranges there between.

In certain aspects, β-sheet peptides (or peptide B) comprise a pluralityof self-assembling peptides. In other aspects the self-assemblingpeptides form a beta-sheet rich fibril. In further aspects, theself-assembling peptide comprises an amino acid sequence of QQKFQFQFEQQ(SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8);KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ IDNO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13);QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE(SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ IDNO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO.19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21);SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (WhereX=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P)(SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO.25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO.27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV(SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO.32); or YTIAALLSPY (SEQ ID NO. 33). In certain aspects, theself-assembling peptide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 to 15, 20, 25, 30, 35, 40, 50, 100, 200, or 500 amino acids inlength, including all values and ranges there between. In certainaspects, more than one self-assembling peptide is present in thecomposition.

In further aspects, the composition or nanostructure may be comprised ina pharmaceutically suitable carrier. For example, the composition may befurther defined as an antigen composition. In other aspects, thecomposition may be in form of a microgel or further defined as microgel.

In further aspects, the composition may not be limited to microgels;particularly the composition may include any 3D structures at amicroscopic scale or a macroscopic scale. For example, the compositionmay be a micro or macro structure with a size, length, diameter of atmost, at least, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0,11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475,480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590,600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710,720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825,830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940,950, 960, 970, 975, 980, 990, 1000 nm, μm, mm, cm or any range derivabletherein. In certain aspects, the composition may be provided as a 3Dcell culture or cell delivery matrix.

There may also be provided methods of providing the compositionsdescribed above. For example, the method may comprise mixing non-β-sheetpeptide tags (or peptide A) and β-sheet peptides (or peptide B). Inparticular aspects, a non-β-sheet peptide tag is attached to a compound.In further aspects, same non-β-sheet peptide tag may be attached to sameor different compounds as active agents. Therefore, there may beprovided a nanofiber complex composition that forms a heterogeneousβ-sheet structure comprising the non-β-sheet peptide tags and β-sheetpeptides comprising different compounds. In further aspects, the methodmay further comprise shaking the mixture, thereby forming any form ofcell culture or cell delivery matrix such as a microgel or amacrostructure.

The methods may involve a precise control of the concentration,temperature, or pH to achieve a nanostructure with a controlled dosage.For example, the pH may be at least, about, or at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or any range derivable therein. Inparticular aspects, the pH may be at about 7. The medium used in themethods may be any aqueous medium, such as phosphate buffered saline. Incertain aspects, the peptide B or β-sheet peptides may be used at orabove its fibrillizing concentration. For example, at least or about1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7.3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0,13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0.19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410,420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520,525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630,640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750,760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870,875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980,990, 1000 mM or μM (or any range derivable therein) of the peptide A (ornon-β-sheet peptides) or peptide B (or β-sheet peptides) may be used inthe methods and compositions described herein.

Certain embodiments are directed to methods of inducing an immuneresponse comprising administering an effective amount of a compositioncomprising one or more antigens. In further aspects, the method may beprovided for treating a patient having or at risk of developing amicrobial infection by administering to the patient an effective amountof a composition described herein. In certain aspects, there may beprovided methods of treating a patient having or at risk of developing acancer, comprising administering to the patient an immunotherapycomprising an effective amount of a composition described herein. Infurther aspects, there may be provided a method of culturing a cell,comprising incubating the cell in a cell culture medium comprising thecomposition described herein, particularly a macroscopic structure or amicrogel composition.

Certain methods and compositions may also be provided for cell delivery,for example, by culturing or suspending cells in the compositionprovided herein for a period, and then delivering the cells to a tissueor a patient or subject. The composition described herein may beformulated into a cell delivery matrix, for example including ECMproteins or protein domains, particularly from the patient's own body.Methods of delivering cells to a subject include delivering the celldelivery composition to particular tissue sites. For instance, thetissue site may include epithelial, connective, skeletal, muscular,glandular, or nervous tissue. A particular tissue site is cardiactissue. In an additional aspect of the method, the subject may be amammal, and in a further aspect the mammal may be a human. One advantageof the cell delivery methods and compositions may be to improve thesurvival and function of the cells when delivered in vivo. In aparticular aspect, at least, at most or about 50, 60, 70, 80, 90, 95,99% or more (or any range derivable therein) of the cells remain viableafter delivery to a tissue site. In a further aspect, the cells aredelivered to the tissue site at a constant rate.

Methods may involve administering to the patient or subject at least orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a pharmaceuticalcomposition or a composition described herein. A dose may be acomposition comprising about, at least about, or at most about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0,11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475,480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590,600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710,720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825,830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940,950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000,4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000,8000, 9000, 10000 milligrams (mg) or micrograms (mcg) or μg/ml ormicrograms/ml or mM or μM (or any range derivable therein) of eachcompound or the total amount of a combination of compounds or thecompositions.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 (A-E): An engineered fusion protein with a β-sheet fibrillizingtail (a “βTail) integrated into nanofibers of β-sheet fibrillizingpeptides in a βTail-dependent manner, without loss of activity. a)Schematic representation of a βTail fusion protein integrating into Q11nanofibers. b-c) βTail underwent slow secondary structural transitionfrom an α-helix to a β-sheet. d-e) A fusion of βTail and GreenFluorescent Protein-UV (βT-GFPuv) integrated into Q11, HK-Q11 and KFE8nanofibers in a βTail-dependent manner. N=3, mean±s.d. for e. *represents p<0.05, ANOVA with Tukey's post-hoc. GFP adapted from PDB1EMA in a.

FIG. 1 (A-F): Engineered fusion proteins with a β-sheet fibrillizingtail that integrated into nanofibers of β-sheet fibrillizing peptides.FIG. 1A) Schematic representation of engineered fusion proteins having aβ-sheet fibrillizing domain integrating into Q11 nanofibers. FIG. 1B-C)The βTail peptide underwent slow secondary structural transition from anα-helix to a β-sheet, whereas Q11 rapidly assembled into β-sheets, and amutated βTail adopted a random coil structure. A fusion of βTail andGreen Fluorescent Protein-UV (βT-GFP) efficiently integrated into Q11nanofibers, whereas a fusion of Q11 and GFP (Q11-GFP) integratedmoderately, and a fusion of GFP and a non-folding βTail mutant(βTmutant-GFP) integrated poorly, as demonstrated by (FIG. 1D) digitalphotographs, and (FIG. 1E) measured by fluorimetry of the supernatantabove sedimented Q11 nanofiber solutions. FIG. 1F) βT-GFP alsointegrated into HK-Q11 and KFE8 nanofibers in a βTail-dependent manner,indicating that co-assembly was not limited to Q11-based nanofibers.N=3, mean±s.d. for e and f. * represents p<0.05, ANOVA with Tukey'spost-hoc. GFP adapted from PDB 1EMA in FIG. 1A.

FIG. 2 (A-D): Fluorescent βTail fusion proteins integrated into Q11nanofibers at a predictable dose without loss of activity. FIG. 2A)βT-GFPuv integrated into Q11 nanofibers over the range of 0.25-1.5 μM ina βTail-dependent manner, as measured by loss of fluorescence from thesupernatant. FIG. 2B) βT-GFPuv integrated into Q11 gels in aβTail-dependent manner without loss of activity. FIG. 2C) ActiveβTail-GFP dose in Q11 gels correlated with βT-GFPuv concentration insolution during assembly. FIG. 2D) Different fluorescent βTail proteinsco-integrated into Q11 gels at a precise molar ratio, as demonstrated bythe close correlation between actual gel color and the predicted color,which was determined by using the protein mole ratio in solution duringassembly as the RGB pixel ratio. N=3, mean±s.d. for FIG. 2A, N=10,mean±s.d. for FIG. 2C.

FIG. 3 (A-D): An enzymatic βTail fusion protein integrated into Q11nanofibers at a predictable dose alone, or with varying amounts ofβTail-GFP, without loss of activity. FIG. 3A) A fusion of βTail and thefungal enzyme cutinase (βT-cut) integrated into Q11 nanofibers over therange of 0.25-1.5 μM in a βTail-dependent manner, as measured by loss ofprotein from the supernatant. FIG. 3B) Q11 nanofibers assembled in thepresence of βT-cut demonstrated cutinase activity, as measured byhydrolysis of p-nitrophenyl butyrate (colorless) to p-nitrophenol(yellow). FIG. 3C) Nanofiber cutinase activity was precisely varied bychanging the concentration of βT-cut present during Q11 assembly. FIG.3D) βT-GFPuv and βT-cut co-integrated into Q11 nanofibers at apredictable ratio without loss of activity, as demonstrated by thedirect correlation between nanofiber fluorescence or cutinase activityand βT-GFPuv or βT-cut concentration, respectively. N=3, mean±s.d.

FIG. 4 (A-E): Q11 and βTail co-assembled into heterogeneous β-sheets.FIG. 4A) TEM identified gold-labeled 2° antibodies bound to Q11nanofibers assembled in the presence of βT-GFPuv and incubated with ananti-GFP 1° antibody, whereas few gold-labeled antibodies bound to Q11assembled in the presence of a βTail peptide and incubated withanti-GFP. FIG. 4B) TEM identified gold-labeled streptavidin bound to Q11nanofibers assembled in the presence of biotinylated βTail, whereas fewgold beads were co-localized with Q11 nanofibers lackingbiotinylated-βTail. FIG. 4C) Tryptophan-terminated βTail (W-βT)integrated into Q11 nanofibers over the range of 25-100 μM in aβTail-dependent manner, as measured by loss of fluorescence from thesupernatant. FIG. 4D-E) βTail secondary structure changed from α-helicalto β-sheet following overnight co-assembly with Q11 at a 1:10 molarratio, whereas βTmutant secondary structure was unchanged in thepresence of a 10-fold molar excess of Q11. N=3, mean±s.d. for c.

FIG. 5 (A-C): Q11 nanofibers bearing a βTail fusion protein acted asself-adjuvanting vaccines, eliciting robust antibodies against proteinantigens in the absence of additional immunostimulatory factors. FIG.5A) Higher anti-GFP total IgG titers were raised by C57BL/6 miceimmunized with βT-GFPuv integrated into Q11 nanofibers when compared totiters raised following immunization with soluble βT-GFPuv. FIG. 5B) Q11nanofibers bearing βT-GFPuv elicited robust serum Ig isotype switchingto predominantly IgG1, whereas soluble βT-GFPuv elicited a more balancedIgG1/IgM profile after primary and booster immunizations. FIG. 5C)Higher anti-cut total IgG titers were raised by C57BL/6 mice immunizedwith βTail-cut integrated into Q11 nanofibers when compared to titersraised following immunization with soluble βTail-cut. N=5, mean±s.d. *represents p<0.05, Student's t-test (FIG. 5A, C), or ANOVA with Tukey'spost-hoc (FIG. 5B), serum titer=1 (dashed line) indicates no detectableantibodies.

FIG. 6 (A-D): Active βTail-GFP (FIG. 6A-B) and βTail-cut (FIG. 6C-D)were recovered from the soluble phase following expression in E. coli,which demonstrated that the βTail domain did not induce aggregation ordisrupt protein folding.

FIG. 7 (A-B): 1 mM Q11 nanofibers were efficiently sedimented bycentrifugation at 12000×g for 5 min, as demonstrated by the formation ofa pellet (FIG. 7A) and the loss of phenylalanine absorbance (X=260 nm)in solution (FIG. 7B). N=3, mean±s.d.

FIG. 8 (A-B): HK-Q11 self-assembled into β-sheet nanofibers in 1×PBS.FIG. 8A) Nanofibers were identified in solutions containing 1 mM HK-Q11in PBS with TEM. FIG. 8B) The fluorescence of thioflavin T, afluorescent dye whose emission increases upon binding to β-sheetfibrils, increased in solutions containing HK-Q11.

FIG. 9: Endotoxin is not a key mediator of adaptive immune responseselicited by nanofibers bearing BT-GFPuv. Total anti-GFP IgG, measured byELISA, raised by TLR4 knock-out C57BL/6 mice 7 weeks after primary andbooster immunization with Q11 and BT-GFPuv (1 mM Q11; primary=90 nmoland booster=80 nmol βTail-GFPuv). N=5, mean±s.e.m.

FIG. 10: Fluorescence emission from βTail-GFPuv, βTail-eGFP, andβTail-dsRED was distinguishable with different fluorescence filtercubes. Exposure times of 0.75 s for βTail-GFPuv, 1.5 s for βTail-eGFP,and 2.5 s for βTail-dsRED enabled visualization of each protein, withoutsignificant background fluorescence from the other proteins.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Certain embodiments are, in part, based on the finding that engineeredfusion proteins having a peptide tag such as a β-sheet fibrillizing tail(a “βTail”) integrated into nanofibers of β-sheet fibrillizingpolypeptides at a tunable dose, without loss of bioactivity. Further,the inventors discovered that multiple different βTail fusion proteinsco-integrated into polypeptide nanofibers, providing biomaterials withmodular and precise composition of bioactive proteins. One aspect alsoinvolves compositions and methods related to the use of protein-bearingnanofibers as self-adjuvanting vaccines, immunogenic compositions orcell culture medium or structure, with the potential of this approachfor creating biomaterials with unique functional properties for variousbiomedical and biotechnological applications, such as therapeutics,tissue engineering, drug delivery, 3-D culture, biosensors, orelectronics.

I. NON-β-SHEET PEPTIDE TAGS

Embodiments involve the use of non-β-sheet peptide tags. The peptidesmay form random coil, α-helical structure or β-sheet structure underdifferent conditions. In particular embodiments, the peptide tags mayform β-sheet structure in the presence of a β-sheet peptide such as aβ-sheet fibrillizing peptide, including self-assembly peptides describedbelow. When isolated or expressed, the peptide tag may form non-β-sheetstructures, such as random coil or α-helical structures (e.g., Pagel2006, incorporated herein by reference).

Such peptide tags may have one or more α-helical motifs or coiled coilmotifs, such as heptad repeats. A coiled coil is a structural motif inproteins. The motif may form a plurality of alpha-helices that arecoiled together like the strands of a rope (dimers and trimers are themost common types). In particular aspects, the peptides may have intactor modified heptad repeats as described below for forming α-helicalstructures or favor forming α-helical structures and also β-sheetforming amino acids or amino acids or amino acids that increase theβ-sheet formation propensity.

The α-helical motifs may contain a repeated pattern, hxxhcxc, ofhydrophobic (h) and charged (c) amino-acid residues, referred to as aheptad repeat. The positions in the heptad repeat may be labeledabcdefg, where a and d are the hydrophobic positions, for example beingoccupied by isoleucine, leucine or valine, b, c, f are likelyhydrophilic or polar residues, and e and g are likely charged resides.Using this nomenclature, each heptad may start with any of a, b, c, d,e, f or g (or a′, b′, c′, d′, e′, f′ or g′), not necessarily a or a′.For example, the heptad repeat may be denoted (a-b-c-d-e-f-g)_(n) or(g-a-b-c-d-e-f)_(n). For example, n may be from about 2, 3, 4, 5, 6, 7,8, 9 to about 10 or more.

In certain embodiments, positions a and/or d may be leucine (L). Infurther embodiments, positions e and g may be a pair of charged resides,such as lysine (K) and glutamic acid (E), or glutamic acid (E) andlysine (K), histidine (H) and glutamic acid (E), glutamic acid (E) andhistidine (H), arginine (R) and glutamic acid (E), glutamic acid (E) andarginine (R), lysine (K) and aspartic acid (D), aspartic acid (D) andlysine (K), histidine (H) and aspartic acid (D), aspartic acid (D) andhistidine (H), arginine (R) and aspartic acid (D) or any other similarlypairs with opposing charges or without opposing charges. In anembodiment, a heptad repeat may have a sequence of (L E K L K S E) ((SEQID NO. 34), (L V V L H S E) (SEQ ID NO. 35), or (L H K L K S E) (SEQ IDNO. 36).

Because positions b, c, and f are solvent exposed, these positions onlyhave a minor impact on α-helical or coiled-coil stability. Thesepositions may be any amino acids, such as glutamic acid (E), lysine (K),histidine (H), serine (S), or valine (V). In particular aspects, thesepositions may comprise one or more β-sheet forming amino acids or aminoacids that increase β-sheet formation propensity compared to itsabsence. Such amino acids may include, but are not limited to glycine,alanine, valine, leucine, and isoleucine, and other non-naturallyoccurring amino acids which may be used in a similar chemical andstructural manner in the peptides.

In further embodiments, the peptide tags may comprise or may not needone or more metal binding sites that may stabilize or disrupt thehelical structure or sheet structure. In particular embodiments, theheptad repeats may have one or more metal binding sites introduced in ani, i+4 geometry that stabilizes the helical structure. For example, twometal binding sites, such as histidine (H), may be incorporated into twoneighboring heptad repeats. Such metal binding sites may be close enoughto each other in the α-helical conformation to help induce or stabilizethe α-helical structure and/or slow down the transition to a β-sheetstructure.

The peptide tags may comprise from 2 to about 200 (e.g. about 3 to about100, such as about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 or anyintermediate ranges) heptad repeats, or particularly 3, 4, 5, 6, 7, 8, 9or 10 heptads. Two or more of the heptads may contain the same repeatingsequence of seven amino acids. Alternatively, each heptad in the peptidetags may be the same or each may be different.

Folding a sequence with this repeating pattern into an alpha-helicalsecondary structure may cause the hydrophobic residues to be presentedas a “stripe” that coils gently around the helix in left-handed fashion,forming an amphipathic structure. The more favorable way for two suchhelices to arrange themselves in the water-filled environment of thecytoplasm is to wrap the hydrophobic strands against each othersandwiched between the hydrophilic amino acids. It is thus the burial ofhydrophobic surfaces that provides the thermodynamic driving force forthe oligomerization. The packing in a coiled-coil interface may beexceptionally tight, with almost complete van der Waals contact betweenthe side chains of the a and d residues.

The α-helices may be parallel or anti-parallel, and may adopt aleft-handed super-coil. Although disfavored, a few right-handedα-helices or coiled coils have also been observed in nature and indesigned proteins.

II. NANOFIBERS

Mixing or co-assembly of β-sheet fibrillizing polypeptides that containa population of peptide tags (particularly referring to a peptide thatcan form both α-helical structures and β-sheet structures underdifferent conditions) with a different population of β-sheet peptides (adifferent peptide) may be used to prepare nanofibers, microgels, orscaffolds in certain aspects of the present invention.

Without limitation one or more β-sheet fibrillizing polypeptidescomprising different molecules such as antigens may be used to preparethe scaffolds, microgels, and nanofibers. The amount of β-sheetfibrillizing polypeptides comprising non-β-sheet peptides and antigenscompared to the β-sheet peptides maybe varied without limitation in thepreparation of the scaffolds, microgels, and nanofibers.

The mixture of first and second peptides may be incubated under anyconditions suitable for forming nanofibers, microgels, or scaffolds. Forexample, the condition may be an aqueous medium, a rocking platform, ora combination thereof. The nanofiber may include any forms ofnanostructures comprising β-sheet secondary structures, such as ananofibril, a nanowire, a nanosurface and a nanosphere.

The self-assembled micelles and nanofibers may be characterized by NOEand FT-IR spectroscopy, circular dichroism; nanofiber fiber networks maybe visualized using transmission electron microscopy (TEM).

III. SELF-ASSEMBLING PEPTIDES

Certain aspects include self-assembling peptides, which may be used inβ-sheet peptides (or peptide B) or polypeptides. Non-limiting examplesof self-assembling peptides have been described in US Patent Publication2012-0282292, which is incorporated herein by reference by its entirety.

As used herein, the term “self-assembling peptide” refers to peptidesthat are able to spontaneously associate and form stable structures. Inone embodiment, a self-assembling peptide comprises an amino acidsequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7);FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO.10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12);QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine)(SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO.16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18);AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20);RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23);WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (SEQID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK(SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30);VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG;KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33) orconservatively modified variants thereof. Self-assembling peptides mayfurther comprise other compounds, for example, immunogenic peptides.

Certain peptides that comprise of alternating hydrophilic andhydrophobic amino acids self-assemble to form an exceedingly stablebeta-sheet macroscopic scaffold (U.S. Pat. Nos. 5,955,343 and 5,670,483,each of which is incorporated herein by reference).

Many self-complementary peptides have identical compositions and length;such as EAK16, KAE16, RAD16, RAE16, and KAD16 have been exemplifiedbelow (Table 1).

TABLE 1 Representative Self-Assembling peptides Name Sequence (n−−>c)SEQ ID NO: Modulus Structure RADA16-I n-RADARADARADARADA-c 20 I βRGDA16-I n-RADARGDARADARGDA-c 37 I r.c. RADA8-I n-RADARADA-c 38 I r.c.RAD16-II n-RARADADARARADADA-c 21 II β RAD8-II n-RARADADA-c 39 II r.c.EAKA16-I n-AEAKAEAKAEAKAEAK-c 17 I β EAKA8-I n-AEAKAEAK-c 40 I r.c.RAEA16-I n-RAEARAEARAEARAEA-c 41 I β RAEA8-I n-RAEARAEA-c 42 I r.c.KADA16-I n-KADAKADAKADAKADA-c 43 I β KADA8-I n-KADAKADA-c 44 I r.c.EAH16-II n-AEAEAHAHAEAEAHAHA-c 45 II β EAH8-II n-AEAEAHAHA-c 46 II r.c.EFK16-II n-FEFEFKFKFEFEFKFK-c 12 II β EFK8-II n-FEFKFEFK-c 47 I βELK16-II n-LELELKLKLELELKLK-c 48 II β ELK8-II n-LELELKLK-c 49 II r.c.EAK16-II n-AEAEAKAKAEAEAKAK-c 18 II β EAK12 n-AEAEAEAEAKAK-c 50 IV/IIα/β EAK8-II n-AEAEAKAK-c 51 II r.c. KAE16-IV n-KAKAKAKAEAEAEAEA-c 52 IVβ EAK16-IV n-AEAEAEAEAKAKAKAK-c 19 IV β RAD16-IV n-RARARARADADADADA-c 53IV β DAR16-IV n-ADADADADARARARAR-c 54 IV α/β DAR16-IV*n-DADADADARARARARA-c 55 IV α/β DAR32-IV n-ADADADADARARARAR-c 56 IV α/βEHK16 n-HEHEHKHKHEHEHKHK-c 57 N/A r.c. EKH8-I n-HEHEHKHK-c 58 N/A r.c.VE20* n-VEVEVEVEVEVEVEVEVEVE-c 59 N/A β RF20* n-RFRFRFRFRFRFRFRFRFRF-c60 N/A β β denotes beta-sheet; α denotes alpha-helix; r.c. denotesrandom coil; N/A denotes not applicable. *Both VE20 and RF20 form abeta-sheet when they are incubated in a solution containing NaCl;however, they do not self-assemble to form macroscopic scaffolds.

The peptides described herein can be chemically synthesized usingstandard chemical synthesis techniques. In some embodiments the peptidesare chemically synthesized by any of a number of fluid or solid phasepeptide synthesis techniques known to those of skill in the art. Solidphase synthesis in which the C-terminal amino acid of the sequence isattached to an insoluble support followed by sequential addition of theremaining amino acids in the sequence is a preferred method for thechemical synthesis of the polypeptides of this invention. Techniques forsolid phase synthesis are well known to those of skill in the art andare described, for example, by Barany and Merrifield (1963) Solid-PhasePeptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.;Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewartet al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill.

IV. COMPOUNDS

Certain embodiments comprise incorporating one or more differentcompounds into a β-sheet assembly via a non-β-sheet peptide thattransitions into a β-sheet structure in the presence of β-sheetpeptides. The compound can be any biocompatible molecules, like a smallmolecule, a drug, a peptide, a lipid, a sugar molecule, or a cell, orany bio-compatible material such as a matrix, a gel, membrane, micelle,or fiber. In particular embodiments, the compound may be an antigen. Asused herein, the term “biocompatible” refers to a substance whichproduces no significant untoward effects when applied to, oradministered to, a given cell or organism according to the methods andamounts described herein.

A. Compounds

In certain embodiments, the compound may be a peptide. A peptide may beany peptide or polypeptide, including, but not be limited to, an enzyme,fluorescent protein, cell-binding domain, cell adhesion domain,extracellular matrix domain, reporter protein, cytokine, antigen,signaling domain, immunomodulating protein, cross-linking protein,hormone, hapten, or a bioactive ligand, such as a peptide, protein, ornucleic acid.

In further embodiments, the compound includes or is substantially in theform of at least one of an organic or inorganic small molecule,clathrate or caged compound, protocell, coacervate, microsphere, Janusparticle, proteinoid, laminate, helical rod, liposome, macroscopic tube,niosome, sphingosome, toroid, vesicular tube, vesicle, small unilamellarvesicle, large unilamellar vesicle, large multilamellar vesicle,multivesicular vesicle, lipid layer, lipid bilayer, micelle, organelle,cell, membrane, nucleic acid, peptide, polypeptide, protein,glycopeptide, glycolipid, lipoprotein, sphingolipid, glycosphingolipid,glycoprotein, peptidoglycan, lipid, carbohydrate, metalloprotein,proteoglycan, chromosome, nucleus, acid, support structure, buffer,protic solvent, aprotic solvent, nitric oxide, nitrous oxide, nitricoxide synthase, amino acid, micelle, polymer, copolymer, monomer,prepolymer, cell receptor, adhesion molecule, cytokine, chemokine,immunoglobulin, antibody, antigen, platelet, extracellular matrix,blood, plasma, cell ligand, zwitterionic material, cationic material,oligonucleotide, nanotube, piloxymer, transfersome, gas, element,contaminant, radioactive particle, hormone, microorganism, bacteria,virus, quantum dot, contrast agent, or any part thereof.

B. Antigens

The term “antigen” may refer to a molecule against which a subject caninitiate a humoral and/or cellular immune response. Antigens can be anytype of biologic molecule including, for example, simple intermediarymetabolites, sugars, lipids, and hormones as well as macromolecules suchas complex carbohydrates, phospholipids, nucleic acids and proteins.

Common categories of antigens include, but are not limited to, viralantigens, bacterial antigens, fungal antigens, protozoa and otherparasitic antigens, tumor antigens, antigens involved in autoimmunedisease, allergy and graft rejection, and other miscellaneous antigens.In certain compositions and methods, the antigen is a peptide.

Viral Antigens.

Examples of viral antigens include, but are not limited to, retroviralantigens such as retroviral antigens from the human immunodeficiencyvirus (HIV) antigens such as gene products of the gag, pol, and envgenes, the Nef protein, reverse transcriptase, and other HIV components;hepatitis viral antigens such as the S, M, and L proteins of hepatitis Bvirus, the pre-S antigen of hepatitis B virus, and other hepatitis,e.g., hepatitis A, B. and C, viral components such as hepatitis C viralRNA; influenza viral antigens such as hemagglutinin and neuraminidaseand other influenza viral components; measles viral antigens such as themeasles virus fusion protein and other measles virus components; rubellaviral antigens such as proteins E1 and E2 and other rubella viruscomponents; rotaviral antigens such as VP7sc and other rotaviralcomponents; cytomegaloviral antigens such as envelope glycoprotein B andother cytomegaloviral antigen components; respiratory syncytial viralantigens such as the RSV fusion protein, the M2 protein and otherrespiratory syncytial viral antigen components; herpes simplex viralantigens such as immediate early proteins, glycoprotein D, and otherherpes simplex viral antigen components; varicella zoster viral antigenssuch as gpI, gpII, and other varicella zoster viral antigen components;Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS 1,NS 1, NS 1-NS2A, 80% E, and other Japanese encephalitis viral antigencomponents; rabies viral antigens such as rabies glycoprotein, rabiesnucleoprotein and other rabies viral antigen components. See FundamentalVirology, Second Edition, e's. Fields, B. N. and Knipe, D. M. (RavenPress, New York, 1991) for additional examples of viral antigens.

Bacterial Antigens.

Bacterial antigens which can be used include, but are not limited to,pertussis bacterial antigens such as pertussis toxin, filamentoushemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and otherpertussis bacterial antigen components; diphtheria bacterial antigenssuch as diphtheria toxin or toxoid and other diphtheria bacterialantigen components; tetanus bacterial antigens such as tetanus toxin ortoxoid and other tetanus bacterial antigen components; streptococcalbacterial antigens such as M proteins and other streptococcal bacterialantigen components; gram-negative bacilli bacterial antigens such aslipopolysaccharides and other gram-negative bacterial antigencomponents; Mycobacterium tuberculosis bacterial antigens such asmycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secretedprotein, antigen 85A and other mycobacterial antigen components;Helicobacter pylori bacterial antigen components; pneumococcal bacterialantigens such as pneumolysin, pneumococcal capsular polysaccharides andother pneumococcal bacterial antigen components; hemophilus influenzabacterial antigens such as capsular polysaccharides and other hemophilusinfluenza bacterial antigen components; anthrax bacterial antigens suchas anthrax protective antigen and other anthrax bacterial antigencomponents; rickettsiae bacterial antigens such as romps and otherrickettsiae bacterial antigen component. Also included with thebacterial antigens described herein are any other bacterial,mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.

Fungal Antigens.

Fungal antigens which can be used in the compositions and methodsinclude, but are not limited to, Candida fungal antigen components;histoplasma fungal antigens such as heat shock protein 60 (HSP60) andother histoplasma fungal antigen components; cryptococcal fungalantigens such as capsular polysaccharides and other cryptococcal fungalantigen components; coccidiodes fungal antigens such as spheruleantigens and other coccidiodes fungal antigen components; and tineafungal antigens such as trichophytin and other coccidiodes fungalantigen components.

Parasite Antigens.

Examples of protozoa and other parasitic antigens include, but are notlimited to, plasmodium falciparum antigens such as merozoite surfaceantigens, sporozoite surface antigens, circumsporozoite antigens,gametocyte/gamete surface antigens, blood-stage antigen pf 1 55/RESA andother plasmodial antigen components; toxoplasma antigens such as SAG-1,p30 and other toxoplasma antigen components; schistosomae antigens suchas glutathione-S-transferase, paramyosin, and other schistosomal antigencomponents; leishmania major and other leishmaniae antigens such asgp63, lipophosphoglycan and its associated protein and other leishmanialantigen components; and trypanosoma cruzi antigens such as the 75-77 kDaantigen, the 56 kDa antigen and other trypanosomal antigen components.

Tumor Antigens.

Tumor antigens which can be used in the compositions and methodsinclude, but are not limited to, telomerase components; multidrugresistance proteins such as P-glycoprotein; MAGE-1, alpha fetoprotein,carcinoembryonic antigen, mutant p53, immunoglobulins of B-cell derivedmalignancies, fusion polypeptides expressed from genes that have beenjuxtaposed by chromosomal translocations, human chorionic gonadotropin,calcitonin, tyrosinase, papillomavirus antigens, gangliosides or othercarbohydrate-containing components of melanoma or other tumor cells. Itis contemplated in certain embodiments that antigens from any type oftumor cell can be used in the compositions and methods described herein.

Antigens Relating to Autoimmunity.

Antigens involved in autoimmune diseases, allergy, and graft rejectioncan be used in certain aspects. For example, an antigen involved in anyone or more of the following autoimmune diseases or disorders can beused in the present invention: diabetes mellitus, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemiclupus erythematosis, autoimmune thyroiditis, dermatitis (includingatopic dermatitis and eczematous dermatitis), psoriasis, Sjogren'sSyndrome, including keratoconjunctivitis sicca secondary to Sjogren'sSyndrome, alopecia areata, allergic responses due to arthropod bitereactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves opthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis. Examples of antigens involved in autoimmune disease includeglutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basicprotein, myelin proteolipid protein, acetylcholine receptor components,thyroglobulin, and the thyroid stimulating hormone (TSH) receptor.Examples of antigens involved in allergy include pollen antigens such asJapanese cedar pollen antigens, ragweed pollen antigens, rye grasspollen antigens, animal derived antigens such as dust mite antigens andfeline antigens, histocompatiblity antigens, and penicillin and othertherapeutic drugs. Examples of antigens involved in graft rejectioninclude antigenic components of the graft to be transplanted into thegraft recipient such as heart, lung, liver, pancreas, kidney, and neuralgraft components. An antigen can also be an altered peptide liganduseful in treating an autoimmune disease.

Examples of miscellaneous antigens which can be can be used in certainaspects include endogenous hormones such as luteinizing hormone,follicular stimulating hormone, testosterone, growth hormone, prolactin,and other hormones, drugs of addiction such as cocaine and heroin, andidiotypic fragments of antigen receptors such as Fab-containing portionsof an anti-leptin receptor antibody.

C. ECM Proteins or Protein Domains

The compounds may include a cell attachment molecule comprising aminoacids, which is an extracellular matrix (ECM) protein, or a peptide thatincludes an ECM protein domain. As known in the art, ECM proteinsprovide structural support to cells and/or attach cells that reside inthe ECM. Molecules on the surface of cells, such as integrins,carbohydrates, and other cell adhesion molecules can interact with ECMproteins to promote cell attachment. Non-limiting exemplary ECM proteinsinclude fibronectin, laminin, collagen, procollagen, elastin,vitronectin, tenascin, entactin, fibrinogen, thrombospondin, osteopontin(bone sialoprotein), osteocalcin, von Willibrand Factor, and activedomains thereof.

ECM protein domains refer to an amino acid sequence found within the ECMprotein that, in itself, provides function according to one or moreproperties of the ECM protein, such as providing structural support tocells and/or for attaching cells. The domain may also be referred to asa “active portion” or “motif.” The peptide that includes an ECM proteindomain can have a “core sequence” of amino acid residues, and optionallyone or more additional amino acid residues that flank (i.e., on theC-terminus, N-terminus, or both) the core sequence. The one or moreadditional amino acids that flank the core sequence can correspond tothe wild type ECM sequence in the relevant region of the protein, or canbe an amino acid(s) that diverges from the wild type sequence (e.g., a“variant amino acid or sequence”). The variant amino acid or sequencecan be one that enhances properties of the peptide, such as providingenhanced ligand interaction, and/or can facilitate formation of thesecond coated layer.

ECM protein domains are known in the art or can be determined usingroutine experimentation by carrying out assays that are commercially ordescribed in a reference. For example, cell attachment assays whichutilize peptides or proteins adhered to plastic or covalentlyimmobilized on a support have been described and can be used todetermine the activity of a desired peptide for promoting attachment ofcells (see, for example, Malinda, K. M., et al. (1999) FASEB J.13:53-62; or Kato, R., et al. (2006) J. Biosci. Bioeng. 101:485-95).

V. PHARMACEUTICAL COMPOSITIONS

Embodiments of the present invention include pharmaceutical compositionscomprising the compositions of nanostructures prepared as above andmethods for using these compositions. These compositions may bepharmaceutical compositions used in preventive care or therapeutics.

For example, the compositions may be immunogenic compositions used forpreventing or ameliorating microbial infections. A particularapplication is prophylaxis for infectious diseases. Exposure to antigensin the nanostructure can create resistance against such diseases or actas a vaccination for various conditions.

In other embodiments, the compositions may be immunogenic compositionsused in immunotherapy, such as a cancer immunotherapy. The antigens usedmay be antigens that are displayed on tumor cells but not healthy cells.Several antigens have been identified as specific to certain types oftumors, such as Caspase-8, MAGE-I, Tyrosinase, HER-2/neu, and MUC-I.With this in mind, nano structures can be used to deliver such antigensto DCs in lymph nodes as a means for activating T cells to attacktumors. The compositions may be administered to a subject or cells invivo or cells in vitro. The cells may be immune cells, such as T cells,B cells, NK cells, or any other immune cells.

As such, certain aspects contemplate vaccines and therapeutics for usein active immunization of subjects. Pharmaceutical compositions such asimmunogenic compositions can include a β-sheet peptide fibril integratedwith non-β-sheet peptide tags coupled to a plurality of antigens,“fibril complex.”

The preparation of pharmaceutical compositions such as vaccinecompositions that contain polypeptide or peptide sequence(s) as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all of which are incorporated herein by reference. Forexample, such pharmaceutical may be prepared as injectables either asliquid solutions or suspensions: solid forms suitable for solution in orsuspension in liquid prior to injection may also be prepared. Thepreparation may also be emulsified.

The active immunogenic ingredient is often mixed with excipients thatare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the pharmaceutical composition such as vaccine maycontain amounts of auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, or adjuvants that enhance the effectivenessof the compositions. In specific embodiments, vaccines are formulatedwith a combination of substances, as described in U.S. Pat. Nos.6,793,923 and 6,733,754, which are incorporated herein by reference.

Pharmaceutical compositions may be conventionally administeredparenterally, by injection, for example, either subcutaneously orintramuscularly. Additional formulations which are suitable for othermodes of administration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkalene glycols or triglycerides: suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10%, preferably about 1%to about 2%. Oral formulations include such normally employed excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 10% to about 95% of active ingredient,preferably about 25% to about 70%.

Upon formulation, compositions may be administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and/or immunogenic. The formulations areeasily administered in a variety of dosage forms. The quantity to beadministered depends on the subject to be treated, including thecapacity of the individual's immune system to synthesize antibodies andthe degree of protection desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are of the order of several hundredmicrograms active ingredient per vaccination. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by subsequentinoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a pharmaceutical composition areapplicable. These are believed to include oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection and the like. The dosage of thepharmaceutical composition will depend on the route of administrationand will vary according to the size and health of the subject.

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects of the present invention involveadministering an effective amount of a composition to a subject. In someembodiments of the present invention, immunogenic compositions may beadministered to the patient to protect against infection by one or moremicrobial pathogens. Additionally, such compounds can be administered incombination with an antibiotic or other known anti-microbial therapy.Such compositions will generally be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The compositions and related methods, particularly administration of apeptide fibril/antigen complex may also be used in combination with theadministration of traditional therapies. These include, but are notlimited to, the administration of antibiotics such as streptomycin,ciprofloxacin, doxycycline, gentamycin, chloramphenicol, trimethoprim,sulfamethoxazole, ampicillin, tetracycline or various combinations ofantibiotics. In cancer immunotherapy, the second therapy may be anycancer treatment such as surgery, chemotherapy, gene therapy, orradiation.

In one aspect, it is contemplated that a peptide fibril/immunogencomposition and/or therapy is used in conjunction with antibacterialtreatment or anticancer therapy. Alternatively, the therapy may precedeor follow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the other agents and/or a proteins isadministered separately, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and antigenic composition would still be able to exert anadvantageously combined effect on the subject. In such instances, it iscontemplated that one may administer both modalities within about 12-24h of each other and, more preferably, within about 6-12 h of each other.In some situations, it may be desirable to extend the time period foradministration significantly, however, where several days (2, 3, 4, 5, 6or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

Various combinations may be employed, for example antibiotic therapy orcancer therapy is “A” and the immunogenic composition is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the immunogenic compositions to a patient/subject willfollow general protocols for the administration of such compounds,taking into account the toxicity, if any. It is expected that thetreatment cycles would be repeated as necessary. It also is contemplatedthat various standard therapies, such as hydration, may be applied incombination with the described therapy.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated. Supplementary active ingredients, such as otheranti-cancer agents, can also be incorporated into the compositions.

The pharmaceutical compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Administration of the compositions will typically be via any commonroute. This includes, but is not limited to oral, nasal, or buccaladministration. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal,or intravenous injection. In certain embodiments, a vaccine compositionmay be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specificallyincorporated by reference). Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

In addition to the compositions formulated for parenteraladministration, such as those for intravenous or intramuscularinjection, other pharmaceutically acceptable forms include, e.g.,tablets or other solids for oral administration; time release capsules;and any other form currently used, including creams, lotions,mouthwashes, inhalants and the like.

The pharmaceutical compositions can be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically,such compositions can be prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for use to preparesolutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and, the preparations can also beemulsified.

Solutions of the pharmaceutical compositions as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered, if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in isotonic NaCl solution andeither added to hypodermoclysis fluid or injected at the proposed siteof infusion, (see for example, Remington's Pharmaceutical Sciences,1990). Some variation in dosage will necessarily occur depending on thecondition of the subject. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

VI. NANOFIBER APPLICATIONS

The nanofiber described herein may be used in many known applicationsemploying nanofibers including, but not limited to, filter applications,computer hard drive applications, and pharmaceutical applications asdescribed above. The nanofiber is useful in a variety of biologicalapplications, including cell culture, tissue culture, and tissueengineering and cell delivery applications.

In one application, a nanofibrillar structure for cell culture andtissue engineering may be fabricated using the nanofiber. In anembodiment, the nanofibrillar structure comprises one or morenanofibers, wherein the nanofibrillar structure is defined by a networkof one or more nanofibers comprising peptide A and peptide B. In anotherembodiment, the nanofibrillar structure comprises one or more nanofibersand a substrate wherein the nanofibrillar structure is defined by anetwork of one or more nanofibers deposited on a surface of thesubstrate. In another application, any cell culture including a growthmedia for cell culture may be prepared using the nanofiber.

In an embodiment, the growth media comprises a matrix of nanofibers inthe form of a mat, roll, or sheet that may be adapted for insertion intoa culture container. In another embodiment, the growth media comprises amatrix of nanofibers that is deposited onto a surface of a culturecontainer or added as a fibrous mesh to the culture container. Inanother application, the nanofiber may be sprayed or spun onto athree-dimensional structure suitable for cell or tissue culture. Theresultant three-dimensional structure is returned to a cell cultureapparatus for continued growth where the electrospun fiber structureserves as a platform for growth of the cells.

In a further application, the nanofibers may be electrospun intononwoven mesh and/or braids for the layered construction ofthree-dimensional matrices to serve as templates for tissueregeneration. In a further application, the nanofibers may be used as acell culture medium in high throughput drug analysis and drugsensitivity analysis to increase the number of cells per well providinghigher signal for detection of cell response. In another furtherapplication, the nanofibers may be used as a cell culture medium in highthroughput drug analysis, drug sensitivity analysis, and othertherapeutic schemes where the nanofibers provide an environment for thecells to more closely mimic the in vivo nature of the cells in an exvivo environment.

The nanofibers may be formed as a cell culture or cell delivery matrix,for example, by including ECM proteins or ECM protein domains. Inparticular aspects, the cell culture or cell delivery matrix maycomprise any degradable, bioabsorbable or non-degradable, biocompatiblepolymer. Exemplary three-dimensional culture or cell delivery materialsinclude polymers and hydrogels comprising collagen, fibrin, chitosan,MATRIGEL™, polyethylene glycol, dextrans including chemicallycrosslinkable or photocrosslinkable dextrans, and the like. In anembodiment, the three-dimensional culture or cell delivery matrixcomprises allogeneic components, autologous components, or bothallogeneic components and autologous components. In an embodiment, thethree-dimensional culture or cell delivery matrix comprises synthetic orsemi-synthetic materials. In an embodiment, the three-dimensionalculture or cell delivery matrix comprises a framework or support, suchas a fibrin-derived scaffold. The term “scaffold” is used herein toinclude a wide variety of three-dimensional frameworks, for example, butnot limited to a mesh, grid, sponge, foam, or the like.

VII. PROTEINACEOUS COMPOSITIONS

In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule and methodsfor preparing and using such compositions. The proteinaceous moleculesmay be used for forming a β-sheet structure or nanofiber or be packagedin the structure as an active compound attached to peptide A ornon-β-sheet peptides.

As used herein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein. For convenience, the terms “protein,” “polypeptide” and“peptide” may be used interchangeably herein.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 275, about 300, about 325, about 350, about 375, about 400,about 425, about 450, about 475, about 500, about 525, about 550, about575, about 600, about 625, about 650, about 675, about 700, about 725,about 750, about 775, about 800, about 825, about 850, about 875, about900, about 925, about 950, about 975, about 1000, about 1100, about1200, about 1300, about 1400, about 1500, about 1750, about 2000, about2250, about 2500 or greater amino molecule residues, and any rangederivable therein.

In some aspects the size of a peptide defined in certain aspects of thepresent invention may comprise, but is not limited to, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues. In otheraspects the size of a peptide may comprise, but is not limited to, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 amino acid residues, or any range derivable therein.In certain embodiments, peptides less than or equal to 20 amino acids,or peptides 6-10 amino acids in length may be used.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 2 below.

TABLE 2 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3- Aminoadipicacid Hyl Hydroxylysine Bala β-alanine, β-Amino- AHyl allo-Hydroxylysinepropionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance which produces no significant untoward effects when appliedto, or administered to, a given cell or organism according to themethods and amounts described herein. Organisms include, but are notlimited to, Such untoward or undesirable effects are those such assignificant toxicity or adverse immunological reactions. In particularembodiments, biocompatible protein, polypeptide or peptide containingcompositions will generally be mammalian proteins or peptides orsynthetic proteins or peptides each essentially free from toxins,pathogens and harmful immunogens.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials.

The nucleotide and protein, polypeptide and peptide sequences forvarious genes have been previously disclosed, and may be found atcomputerized databases known to those of ordinary skill in the art. Onesuch database is the National Center for Biotechnology Information'sGenbank and GenPept databases (available at www.ncbi.nlm.nih.gov/). Thecoding regions for these known genes may be amplified and/or expressedusing the techniques disclosed herein or as would be known to those ofordinary skill in the art. Alternatively, various commercialpreparations of proteins, polypeptides and peptides are known to thoseof skill in the art.

B. Purification or Isolation

In certain embodiments a protein or peptide or a composition comprisingsuch a protein or peptide may be isolated or purified. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the homogenization and crudefractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography and isoelectric focusing.An example of receptor protein purification by affinity chromatographyis disclosed in U.S. Pat. No. 5,206,347, the entire text of which isincorporated herein by reference. A particularly efficient method ofpurifying peptides is fast performance liquid chromatography (FPLC) oreven high performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present invention, means that the peptide, polypeptide orprotein has a level of purity where the peptide, polypeptide or proteinis substantially free from other proteins and biological components. Forexample, a purified peptide, polypeptide or protein will often besufficiently free of other protein components so that degradativesequencing may be performed successfully.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A particular methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides may be subjected to fractionation to removevarious other components from the composition. Various techniquessuitable for use in protein purification are well known to those ofskill in the art. These include, for example, precipitation withammonium sulphate, PEG, antibodies and the like, or by heatdenaturation, followed by: centrifugation; chromatography steps such asion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. Certain aspects of the present invention provide DNA sequences forthe specific proteins, and any fusion protein purification method may bepracticed. However, given many DNA and proteins are known, or may beidentified and amplified using the methods described herein, anypurification method can now be employed.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that itself does not adsorb molecules toany significant extent and that has a broad range of chemical, physicaland thermal stability. The ligand should be coupled in such a way as tonot affect its binding properties. The ligand should also providerelatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand.

C. Fusion Proteins

Other embodiments of protein conjugates concern fusion proteins. Thesemolecules generally have all or a substantial portion of an antigenicpeptide, linked at the N- or C-terminus, to all or a portion of a secondpolypeptide or protein. For example, fusions may employ leader sequencesfrom other species to permit the recombinant expression of a protein ina heterologous host. Another useful fusion includes the addition of animmunologically active domain, such as an antibody epitope, to, forexample, facilitate purification of the fusion protein. Inclusion of acleavage site at or near the fusion junction will facilitate removal ofthe extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

In particular embodiments, the fusion proteins comprise a peptide tagattached to an antigenic protein or peptide. Examples of proteins orpeptides that may be incorporated into a fusion protein includecytostatic proteins, cytocidal proteins, pro-apoptosis agents,anti-angiogenic agents, hormones, cytokines, growth factors, peptidedrugs, antibodies, Fab fragments antibodies, antigens, receptorproteins, enzymes, lectins, MHC proteins, cell adhesion proteins andbinding proteins. These examples are not meant to be limiting and it iscontemplated that within the scope of the present invention virtuallyany protein or peptide could be incorporated into a fusion proteincomprising a peptide tag. Methods of generating fusion proteins are wellknown to those of skill in the art. Such proteins can be produced, forexample, by chemical attachment using bifunctional cross-linkingreagents, by de novo synthesis of the complete fusion protein, or byattachment of a DNA sequence encoding a peptide tag to a DNA sequenceencoding the second peptide or protein, followed by expression of theintact fusion protein.

D. Synthetic Peptides

Because of their relatively small size, the peptides in certain aspectscan be synthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, 1984; Tam et al., 1983; Merrifield, 1986;Barany and Merrifield, 1979, each incorporated herein by reference.Short peptide sequences, usually from about 6 up to about 35 to 50 aminoacids, can be readily synthesized by such methods. Alternatively,recombinant DNA technology may be employed wherein a nucleotide sequencewhich encodes a peptide is inserted into an expression vector,transformed or transfected into an appropriate host cell, and cultivatedunder conditions suitable for expression.

E. Linkers/Coupling Agents

If desired, the compound or peptides of interest may be joined via abiologically-releasable bond, such as a selectively-cleavable linker oramino acid sequence. For example, peptide linkers that include acleavage site for an enzyme preferentially located or active within atumor environment are contemplated. Exemplary forms of such peptidelinkers are those that are cleaved by urokinase, plasmin, thrombin,Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase,gelatinase, or stromelysin.

Additionally, while numerous types of disulfide-bond containing linkersare known which can successfully be employed to conjugate moieties,certain linkers will generally be preferred over other linkers, based ondiffering pharmacologic characteristics and capabilities. For example,linkers that contain a disulfide bond that is sterically “hindered” areto be preferred, due to their greater stability in vivo, thus preventingrelease of the moiety prior to binding at the site of action.

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art can be used to combine to components oragents of the present, such as, for example, antibody-antigeninteraction, avidin biotin linkages, amide linkages, ester linkages,thioester linkages, ether linkages, thioether linkages, phosphoesterlinkages, phosphoramide linkages, anhydride linkages, disulfidelinkages, ionic and hydrophobic interactions, bispecific antibodies andantibody fragments, or combinations thereof.

Cross-linking reagents may be used to form molecular bridges that tietogether functional groups of two different molecules, e.g., astabilizing and coagulating agent. However, it is contemplated thatdimers or multimers of the same analog can be made or that heteromericcomplexes comprised of different analogs can be created. To link twodifferent compounds in a step-wise manner, hetero-bifunctionalcross-linkers can be used that eliminate unwanted homopolymer formation.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Supramolecular Assemblies with Precise Composition of MultipleDifferent Bioactive Protein Ligands

An engineered variant of GFP having a β-sheet fibrillizing tail (i.e. a“βTail”) integrated into nanofibers of β-sheet fibrillizing polypeptidesin a βTail-dependent manner without loss of activity (FIG. 1). The βTailsequence, LKVELEKLKSELVVLHSELHKLKSEL [SEQ ID NO. 3], was chosen becauseit underwent slow transition to a β-sheet (FIG. 1B-C) (Pagel, 2008).This property enabled the expression and subsequent assembly of βTailfusion proteins into nanofibers, as demonstrated by the recovery ofactive fluorescent and enzymatic βTail fusion proteins from the solublephase following expression in E. coli (FIG. 6). This was in starkcontrast to the rapid misfolding and aggregation of GFP fused to aβ-sheet fibrillizing amyloid-β domain previously reported (Kim 2006), orthe behavior of Q11-tagged fusion proteins, which could be expressed,but which did not assemble into nanofibers efficiently (FIG. 1D-E). Q11peptide nanofibers were efficiently sedimented by centrifugation (FIG.7), and >80% of βTail-GFP in solution sedimented with Q11 nanofibers(FIGS. 1D-E). On the other hand, GFP was primarily retained in thesupernatant when the βTail domain was mutated to a non-folding variant,or when Q11 was used as the fusion tag (FIGS. 1D-E). In addition,βTail-GFP was retained in the supernatant when solutions lacked Q11peptides (FIG. 1D), which demonstrated that βTail-GFP did not undergosignificant self-assembly during the incubation period. βTail-GFP alsoefficiently integrated into nanofibers of HK-Q11 and KFE8, two otherβ-sheet fibrillizing polypeptides (FIG. 8) (Marini 2002), and this wasagain dependent on the βTail domain (FIG. 1E). On the other hand, bothβTail-GFP and its mutated counterpart bound with similar efficiency tonanofibers of the β-sheet fibrillizing polypeptide RADA16 (data notshown) (Zhang 1995), which suggested that GFP interactednon-specifically with RADA16 nanofibers. Together, these observationsindicated that βTail fusion proteins can integrate into β-sheetfibrillizing polypeptide nanofibers via the βTail domain, although thespecificity of this interaction may be dependent on the primary sequenceof the β-sheet fibrillizing polypeptide.

βTail-GFP integrated into Q11 nanofibers at a precise dose alone, or incombination with other fluorescent βTail fusion proteins, resulting insupramolecular biomaterials with precise composition of multipledifferent bioactive protein ligands (FIG. 2). βTail-GFP concentration inQ11 nanofibers sedimented by centrifugation correlated with βTail-GFPconcentration in solution during nanofiber assembly (FIG. 2A). This wasagain dependent on the βTail domain and the presence of Q11, since GFPfused to a mutated βTail domain, as well as GFP lacking a fusion domain,were retained in the supernatant following centrifugation.

In addition, βTail-GFP was not sedimented by centrifugation in theabsence of Q11 at any concentration tested, which demonstrated thatβTail fusion proteins do not appreciably self-assemble over thisconcentration range (FIG. 2A). Q11 microgels were fluorescent whenassembled in the presence of βTail-GFP, and this was also dependent onthe βTail domain (FIG. 2B). βTail-GFP fluorescence was not perturbedwhen integrated into Q11 nanofibers, which was consistent with previousreports of GFP integrated into other polypeptide nanofibers (Baxa 2002),and GFP fluorescence could be dosed into Q11-based biomaterials byvarying GFP concentration in solution during nanofiber assembly (FIG.2C).

Similarly, fusions of βTail-GFPuv, βTail-enhanced GFP (βTail-eGFP), andβTail-dsRED monomer (βTail-dsRED) co-integrated into Q11 nanofibers atpredictable concentrations, which correlated with the molar ratio ofproteins present in solution during nanofiber assembly (FIG. 2D). Inparticular, a mixture consisting of an equimolar ratio of βTail-GFPuv,βTail-dsRED, and βTail-eGFP provided a gray Q11 microgel, which closelymatched the predicted gray color for an RGB image with 33% red, 33%green, and 33% blue pixels. Varying the mole ratio of βTail-GFPuv,βTail-eGFP, and βTail-dsRED present during co-assembly enabledfine-tuning of Q11 microgel color, as demonstrated by microgels withcolors ranging from pink to orange to teal that closely matched thepredicted color (FIG. 2D). On the other hand, when a non-foldingβTmutant-GFPuv variant was added in place of βTail-GFPuv in an equimolarmixture, the resulting microgels did not match the predicted gray color(FIG. 2D).

Together, these results demonstrated that multiple different βTailfusion proteins with a similar tertiary structure co-integrated intosynthetic polypeptide nanofibers at a precise dose and in aβTail-dependent manner, without significantly perturbing proteinbioactivity.

Polypeptide nanofibers bearing a single protein antigen elicit robust,protein-reactive antibodies in the absence of additionalimmunostimulatory molecules (Hudalla 2013), and based on the data inthis Example, this may be extended to include nanofibers bearingmultiple different protein antigens would provide the basis for newsubunit vaccines that approach the broad-spectrum, multi-antigenimmunity conferred by live or attenuated pathogens

It was demonstrated that a recombinant fusion of βTail and the fungalenzyme cutinase (βTail-cut) integrated into Q11 nanofibers without lossof activity to highlight the versatility of this approach (FIGS. 3A-3D),and because GFP and its homologs are relatively robust proteins that arebroadly amenable to expression as recombinant fusions. βTail-cutconcentration in Q11 nanofibers sedimented by centrifugation correlatedwith βTail-cut concentration in solution during Q11 assembly, and thiswas dependent on the presence of Q11 (FIG. 3A). Q11 nanofibersdemonstrated cutinase activity when assembled in the presence of βT-cut,as measured by hydrolysis of p-nitrophenyl butyrate to p-nitrophenol(FIG. 3B) (Kolattukudy 1981), and cutinase activity could be dosed intoQ11 nanofibers by simply varying βTail-cut concentration in solutionduring Q11 assembly (FIG. 3C). Notably, βTail-cut and βTail-GFPco-integrated into Q11 nanofibers at a predictable dose, without loss ofactivity, by varying the molar ratio of the proteins present during Q11assembly (FIG. 3D). Importantly, this demonstrated that two distinctproteins having vastly different amino acid composition, tertiarystructure, and bioactivity can be co-integrated into polypeptidenanofibers via the βTail fusion approach, suggesting the widespreadpotential of this system for creating biomaterials with new functionalproperties.

Using TEM, the co-assembly of Q11 and βTail into heterogeneous β-sheetnanofibers was characterized in more detail (FIG. 4), based on theobservations that Q11 and βTail self-assembled into β-sheet richnanofibers independently (FIGS. 1B-C) (Pagel 2008; Collier 2003, each ofwhich is incorporated herein for reference), and that βTail fusionproteins specifically interacted with Q11 nanofibers (FIG. 1-2). TEMidentified a gold-labeled 2° antibody co-localized with Q11 nanofibersassembled in the presence of βT-GFPuv and then incubated with ananti-GFP antibody, whereas few gold-labeled 2° antibodies wereassociated with Q11 assembled in the presence of βT and then incubatedwith anti-GFP (FIG. 4A). These results further supported earlierobservations that βT-GFP integrated into Q11 nanofibers (FIGS. 1D-E;FIG. 2A-B). TEM also identified gold-labeled streptavidin co-localizedwith Q11 nanofibers assembled in the presence of biotinylated-βTail,whereas gold-labeled streptavidin failed to bind to Q11 nanofibersassembled in the absence of biotinylated-βTail (FIG. 4B), demonstratingthat βTail was integrated into Q11 nanofibers even in the absence of alarge protein domain. Tryptophan-terminated βTail (W-βT) could be dosedinto Q11 nanofibers at a precise concentration, as measured by loss oftryptophan fluorescence from the supernatant following centrifugation,and this was dependent on both the βTail sequence and the presence ofQ11 (FIG. 4C). Taken together, these observations demonstrated thatβTail polypeptides integrated into Q11 nanofibers in a sequence-specificmanner, regardless of whether they were fused to a protein, a peptide,or a small molecule. This is noteworthy because it suggests that theβTail domain may be broadly useful for co-integrating variousbiologically active ligands into β-sheet nanofibers using the samesimple mixing strategies commonly used to create multi-componentnanofibers from different synthetic polypeptides having an identicalself-assembling domain (Jung 2011; Gasiorowski, 2011).

Circular dichroism (CD) was used to further characterize Q11 and βTailco-assembly into heterogeneous β-sheets. βTail adopted an α-helical 2°structure in the absence of Q11, and transitioned to a predominantlyβ-sheet 2° structure in the presence of a 10-fold molar excess of Q11(FIG. 4D). On the other hand, a non-folding, mutated βTail variantadopted a random coil conformation alone, as well as in the presence ofexcess Q11 (FIG. 4E). These observations were consistent with previousreports demonstrating significant changes to CD spectra followingco-assembly of two different polypeptides into heterogeneous β-sheets(Takahashi 2002; Bothner 2003), and suggested that Q11 and βTailco-assembled into heterogeneous β-sheet nanofibers. Notably, theprotein-bearing nanofibers described above (FIG. 1-3) are likely alsoheterogeneous β-sheets, since they were prepared at a much greaterQ11:βTail molar ratio (1000:1) than Q11:βTail solutions characterized byCD (10:1). Therefore, this approach provides a simple route to directlyintegrate one or more protein ligands at a precise concentration intoβ-sheet polypeptide nanofibers via non-covalent co-assembly, therebyeliminating the need rely on affinity tags or covalent capture ligandsto immobilize protein ligands onto mature nanofibers at precise doses(Hudalla 2013; Sangiambut 2013).

Here, the ability of Q11 nanofibers bearing βTail-GFP to act asself-adjuvanting vaccines provided an initial demonstration of thebiomedical potential of these materials (FIGS. 5A-5C). C57BL/6 miceimmunized with βTail-GFP bearing nanofibers raised more circulatinganti-GFP antibodies compared to mice immunized with soluble βTail-GFP(FIG. 5A). In addition, mice immunized with βTail-GFP bearing nanofibersunderwent robust isotype switching towards predominantly IgG1 followingprimary and booster immunizations, whereas mice immunized with solubleβTail-GFP demonstrated a more balanced IgM/IgG1 isotype profile (FIG.5B). C57BL/6 mice immunized with βTail-cut bearing nanofibers alsoraised more circulating anti-cut antibodies than mice immunized withsoluble βTail-cut (FIG. 5C). These results demonstrated that polypeptidenanofibers bearing an integrated protein antigen act as vaccines toelicit adaptive immune responses against an antigen, and were consistentwith a recent report demonstrating that Q11 nanofibers bearingcovalently conjugated GFP elicited robust anti-GFP antibodies,²⁴ furthersuggesting the potential of these protein-bearing nanofibers forcreating self-adjuvanting supramolecular vaccines. Because the βTailproteins were expressed in E. coli, significant steps were taken toensure that the endotoxin content of all vaccines was ≦1 EU/mL, which isthe maximum allowable dose for pre-clinical vaccines.⁴³ However, tofurther rule out the role of endotoxin in the observed immune responses,C57BL/6 mice lacking expression of Toll-like receptor-4 with Q11nanofibers bearing βTail-GFP were immunized. These mice also raised highconcentrations of circulating anti-GFP antibodies following immunizationwith βTail-GFP bearing nanofibers (FIG. 9), which demonstrated thatendotoxin contaminants are not a key mediator of the observed responses.Together, these observations indicated that nanofibers bearing βTailfusion proteins acted as self-adjuvanting vaccines in the absence ofadditional immunostimulatory molecules, similar to nanofibers having acovalently conjugated protein,²⁴ suggesting that the nanofiber itselfacts as the vaccine adjuvant to boost adaptive immune responses elicitedagainst protein antigens.

Taken together, the results demonstrated that engineered fusion proteinshaving a β-sheet fibrillizing tail integrated into nanofibers of β-sheetfibrillizing polypeptides. Fluorescent and enzymatic proteins, for whichbioactivity and tertiary structure are directly linked, were expressedas soluble, bioactive βTail fusion proteins and retained their nativebioactivity when integrated into polypeptide nanofibers, which suggestedthe broad potential of this approach for integrating proteins withdiverse biological or chemical activities into supramolecularassemblies. Varying the concentration of βTail fusion proteins presentin solution during Q11 self-assembly provided precise control overprotein content in the nanofibers, similar to approaches to immobilizefolded proteins onto mature nanofibers via affinity tags or covalentcapture ligands (Hudalla 2013; Sangiambut 2013). Notably, it was alsodemonstrated that multiple different βTail fusion proteins co-integratedinto polypeptide nanofibers at a precise dose, resulting insupramolecular biomaterials displaying modular and tunable activityrelated to each protein. Although supramolecular assemblies with preciseprotein composition have been demonstrated previously (Brodin 2012; King2012; Sinclair 2011), this could be the first demonstration ofintegrating multiple different protein ligands at precisely defineddoses into supramolecular biomaterials. Importantly, this suggested theenormous potential of this general approach for creating novelbiomaterials with unprecedented functional properties, which approachthe biomolecular complexity inherent to supramolecular assemblies thatgovern diverse processes throughout living systems.

Here, it was observed that nanofibers with integrated βTail proteinselicited robust anti-protein antibodies in the absence of additionalimmunostimulatory molecules, suggesting the potential of these materialsfor developing new subunit vaccines for disease prophylaxis orimmunotherapy. Creating efficacious multi-antigen vaccines is a keyobjective in modern vaccinology because of the potential forsimultaneously raising broad-spectrum immunity against differentpathogens or pathogen serotypes, while minimizing the number ofimmunization administered. An important consideration in the design ofmulti-antigen vaccines is the potential for “antigenic dominance”, inwhich adaptive immune responses are selectively elicited against oneantigen in a co-administered antigen mixture. The ability to preciselytitrate two or more different antigens into polypeptide nanofiberadjuvants is likely to greatly improve efforts to minimize antigenicdominance during optimization of multi-antigen vaccine formulations,when compared to poorly controlled, non-specific antigen adsorption ontoconventional adjuvants, such as aluminum hydroxide microparticles.However, in light of the general nature and unprecedented versatilityafforded by this approach, it was contemplated that βTail fusionproteins, or analogous strategies, will be widely used to createsupramolecular biomaterials for diverse medical and technologicalapplications, including drug delivery, synthetic extracellular matricesfor tissue engineering and 3-D culture, biosensors, and beyond.

Example 2 Materials and Methods

Peptide Synthesis.

Dimethylformamide, diethyl ether, trifluoroacetic acid (TFA), anddichloromethane were purchased from Fisher Scientific. Piperidine,p-nitrophenyl butyrate, and acetic acid were purchased fromSigma-Aldrich. All amino acids and Rink Amide AM resin were purchasedfrom Novabiochem. The β-sheet fibrillizing polypeptides Q11(QQKFQFQFEQQ) [SEQ ID NO. 6] (Kolattukudy 1981), HK-Q11 (QQKFQFQFHQQ)[SEQ ID. 7] (FIG. 8), KFE8 (FKFEFKFE) [SEQ ID NO. 8] (Marini 2002), andRADA16 (RADARADARADARADA) [SEQ ID NO. 20] (Zhang 1995), as well as βTailpeptides, βTail (MALKVELEKLKSELVVLHSELHKLKSEL [SEQ ID NO. 61]), W-βTail(WGSGSMALKVELEKLKSELVVLHSELHKLKSEL) [SEQ ID NO. 62], and βTail mutant(GKPEGEKPKSEGGPGHSEGHKPKSEG) [SEQ ID NO. 63], were synthesized using astandard Fmoc solid-phase peptide synthesis protocol involvingDIEA/HOBt/HBTU activation. Biotinylated-βTail was synthesized byreacting resin-bound NH₂—SGSG-βTail with Biotin-ONp (Novabiochem) at a2.5:1 molar excess of biotin to primary amines in DMF overnight.Peptides were cleaved from the resin using 95% TFA/2.5% TIS/2.5% DI H2Oand precipitated from the TFA cocktail using cold diethyl ether.Peptides were collected by centrifugation and washed five times inether. The resulting peptide pellet was dried over vacuum, dissolved indeionized water, frozen, and lyophilized to dryness. Peptides werepurified using a Varian ProStar HPLC system, Grace-Vydac C18reverse-phase columns, and water-acetonitrile+0.1% TFA gradients togreater than 90% purity. Peptide molecular weight was verified usingMALDI-TOF-MS on an Applied Biosystems Voyager system 6187 withα-cyano-4-hydroxycinnamic acid as the matrix.

Fusion Protein Construction.

The region encoding cutinase within a pET-21d vector containing therecombinant genetic fusion cutinase-(Gly-Ser linker)-GFPuv-His₆(described previously in Hudalla 2013) was excised from the gene bydigestion with NcoI and BamHI restriction enzymes. Complimentaryoligonucleotides encoding βTail or βTmutant with an NcoI site at the 5′end and BamHI at the 3′ end were synthesized by IDT DNA technologies(Iowa), annealed by heating to 95° C. for 3 min then allowing theheating block to cool to room temperature over 60 min, and digested withNcoI and BamHI restriction enzymes. Digested plasmid andoligonucleotides were purified by electrophoresis on a 1% agarose gel,followed by isolation with the QIAquick Gel Extraction kit (Qiagen).Oligonucleotides and plasmid were mixed at a 6:1 molar ratio in T4 DNAligase buffer (New England Biolabs) heated at 45° C. for 1 min, spikedwith 400 units of T4 DNA ligase, and incubated overnight at 18° C.Plasmids were then transformed into OneShot Top10 E. coli (Invitrogen)and grown on LB-agar plates with 100 ug/mL ampicillin. For expression,plasmids were isolated from Top10 E. coli using the QIAquick Miniprepkit (Qiagen), transformed into Origami B (DE3) E. coli, and grown onLB-agar plates with 100 ug/mL ampicillin and 50 ug/mL kanamycin. ApET-21d vector encoding βTail-cut was prepared using a similar method,except a nucleic acid sequence encoding cutinase was amplified out ofthe vector encoding cutinase-(Gly-Ser linker)-GFP using primers having a5′ BglII end and a 3′ XhoI end. This gene and the pET-21d vectorencoding βTail-(Gly-Ser linker)-GFP were digested with BglII and XhoI,purified, ligated together, and transformed into E. coli. Vectorsencoding βTail-eGFP and βTail-dsRED were synthesized and subcloned intopET-21D by Genscript (New Jersey, USA). The sequence of each βTailfusion was confirmed by sequencing performed at the University ofChicago Sequencing Facility (see supplemental materials for fusionprotein nucleotide and amino acid sequences).

Fusion Protein Expression and Purification.

Ten milliliters of 2XTY media with 100 μg/mL ampicillin and 50 μg/mLkanamycin A was inoculated with E. coli and maintained overnight at 37°C., 220 rpm. The 10 mL culture was subcultured into 1 L 2XTY with 100μg/mL ampicillin and 50 μg/mL kanamycin A and maintained at 37° C., 220rpm until an optical density of 0.6 at λ=600 nm was reached. Proteinexpression was then induced by adding 0.5 mM IPTG to the culture andmaintaining the culture at 37° C., 100 rpm for 4 h. Cells were collectedby centrifugation, washed, and lysed into 1×PBS containing 1× BugBusterProtein Extraction Reagent (Novagen, CA), 1 eComplete protease inhibitortablet (Santa Cruz Biotechnology), 300 units DNAse I from bovinepancrease (Sigma-Aldrich, MO), and 100 μg lysozyme for 20 min at roomtemperature. The lysis buffer was cleared by centrifugation andHis6-tagged βTail fusion proteins were purified from the supernatantusing metal-affinity chromatography on HisPur cobalt resin (ThermoScientific, IL). Protein was eluted from the column withimidazole-containing buffers and concentrated into 1×PBS usingcentrifugal filter units with a 10,000 DA MWCO (Millipore). Endotoxincontent was reduced using Triton X-114 cloud-point precipitation,according to previously reported methods (Hudalla 2013). Briefly, TritonX-114 was added to proteins at a 1:10 (v/v) ratio at 4° C., thesesolutions were maintained on ice for 20 min, and heated to 37° C. for 10min. Endotoxin-loaded Triton X-114 micelles were then removed bycentrifugation at 5000×g, and the process was repeated two additionaltimes.

Nanofiber Assembly.

Lyophilized Q11 was dissolved in deionized water at a finalconcentration of 10 mM by vortexing for 5 min. Aqueous Q11 solutionswere diluted 10-fold with 1×PBS containing GFP (Vector Labs cat#MB-0752), βTmutant-GFP, or one or more βTail fusion proteins at a totalprotein concentration between 0.25-1.5 μM, or tryptophan-terminatedβTail (W-βTail) at a concentration between 10-100 μM. These solutionswere then incubated under static conditions for nanofiber assembly, oron a LabNet Rocker 35 rocker table (speed setting 4) (New Jersey) toinduce microgel formation.

Characterizing Protein Integration into Nanofibers.

Q11 nanofibers assembled overnight in the presence of βTail fusionproteins were sedimented by centrifugation at 12000×g for 5 min. Thesupernatant was removed and analyzed for βTail-GFP, βTmutant-GFP, GFP,or W-βTail content by measuring fluorescence emission with a SpectraMaxM5 (excitation 395 nm/emission 503 nm for GFP; excitation 280nm/emission 325 nm for W-βTail, glass-bottom 96-well plates were usedfor W-βTail fluorescence measurements), and converting emissionintensity to protein concentration using GFP or W-βTail fluorescencestandards. Additionally, nanofibers were resuspended in fresh 1×PBS, andnanofiber fluorescence was determined using a SpectraMax M5 platereader. The μBCA assay kit (Pierce) was used according to themanufacturer's instructions to determine βTail-cut concentration in thesupernatant. Protein concentration in the nanofibers was reported as thedifference between the protein concentration in solution duringnanofiber assembly and the protein concentration in the supernatantafter centrifugation.

Fluorescence Microscopy.

Q11 microgel fluorescence was analyzed using a Zeiss Axioscope invertedepifluorescent microscope. Dapi filters were used to visualizeβTail-GFPuv; FITC filters were used to visualize βTail-eGFP; and TRITCfilters were used to visualize βTail-dsRED. Fluorescence emission of anyprotein with the inappropriate filter (e.g. βTail-GFPuv with FITC) wasnegligible (FIG. 5). Due to differences in the quantum efficiency ofeach protein, exposure time was adjusted until the grayscale imageintensity of microgels containing 0.33 μM βTail-GFP, βTail-eGFP, orβTail-dsRED alone was similar (e.g. 0.75 sec for βTail-GFP, 1.5 sec forβTail-eGFP, and 2.5 sec for βTail-dsRED). These exposure times were thenused to collect grayscale images of microgels formed from solutionscontaining βTail-GFP, βTail-eGFP, and βTail-dsRED at different molarratios with each fluorophore filter cube. Grayscale microgel images werepseudocolored red, green, or blue according to the filter cube set used,and then merged using ImageJ software (NIH).

Cutinase Activity.

Cutinase hydrolyzes the colorless molecule p-nitrophenyl butyrate to theyellow molecule p-nitrophenol, which allows for colorimetric analysis ofcutianse activity (Kolattukudy 1981). Cutinase activity of Q11nanofibers assembled in the presence of 0.25-1.5 μM βTail-cut wasanalyzed by adding 1 μL of 0.1 M p-nitrophenyl butyrate (Sigma-Aldrich)in dimethyl sulfoxide (Fisher Scientific) to 100 μL of nanofibers in1×PBS and measuring p-nitrophenol absorbance at 405 nm for 3 min using aSpectraMax M5 plate reader. The initial velocity, v_(o), ofp-nitrophenyl butyrate hydrolysis to p-nitrophenol was calculated fromthe linear portion of a plot of A405 nm versus time.

Transmission Electron Microscopy.

TEM was performed to visualize βTail-GFP or biotinylated-βTailintegration into Q11 nanofibers using previously reported methods(Gasiorowski 2011), with minimal modifications. 1 mM Q11 was assembledin the presence of 1 μM βTail or 1 μM βTail-GFP, or 500 μM Q11 wasassembled alone, or in the presence of 50 μM biotinylated-βTail, usingmethods outlined above. Nanofiber solutions were diluted to 0.25 mM Q11with 1×PBS, and nanofibers were adsorbed onto 200 mesh lacey carbongrids, blocked with 2% acetylated bovine serum albumin (BSA)/0.1% coldwater fish skin gelatin, and placed onto a series of droplets containing(1) monoclonal mouse anti-GFP antibody (Santa Cruz Biotechnology cat.#sc-9996, 1:4 in PBS), (2) goat anti-mouse IgG-15 nm gold particles (EMScat. #25133), and streptavidin-5 nm gold particles (Invitrogen cat. #A32360, all particles diluted 1:4 in PBS), and (3) 1% uranyl acetate inwater. Triplicate PBS washes were performed between staining steps.Grids were analyzed with an FEI Tecnai F30 TEM.

Circular Dichroism.

Circular dichroism was performed using an Aviv303 circular dichroismspectrometer in the University of Chicago Biophysics Core. Solutionscontaining 25 μM βTail or βTmutant, 250 μM Q11, 25 μM βTail plus 250 μMQ11, or 25 μM βTmutant plus 250 μM Q11 in 1× phosphate buffer plus 120mM potassium fluoride were analyzed after overnight assembly understatic conditions at room temperature. Each sample was analyzed 3 times,and the averaged spectrum was reported.

Immunizations.

Q11 microgels bearing βTail-GFP or βTail-cut were prepared as describedabove, except the materials were incubated at 4 deg C, rather than roomtemperature. GFP content in the microgels was analyzed fluorimetricallyas described above, and the concentration of GFP in the microgels wasused as the basis for βTail-GFP dose in the PBS group. Similarly,cutinase content in the microgels was analyzed with the μBCA assay kit(Pierce), according to the manufacturer's instructions, and βTail-cutdose in the microgels was used as the basis for βTail-cut dose in PBSgroup. Endotoxin content of all vaccines was analyzed with the LimulusAmoebocyte Lysate assay kit (Lonza) according to the manufacturer'sinstructions immediately before immunization, and all materials werebelow the upper limit of 1 EU/mL. Female C57BL/6 mice (6-8 weeks old,Taconic Farms, IN) were each given two 50 μL subcutaneous injectionsnear the shoulder blades for each primary and booster immunization,similar to previously reported methods (Hudalla 2013). The GFP dosingregimen was: 90 nmol βTail-GFP with or without 1 mM Q11 at day 0, and 80nmol βTail-GFP with or without 1 mM Q11 at day 31; the cutianse dosingregimen was: 108 nmol βTail-cut with or without 1 mM Q11 at days 0, 28,and 63. Blood was collected weekly via the submandibular vein.Institutional guidelines for the care and use of laboratory animals werestrictly followed under a protocol approved by the University ofChicago's Institutional Animal Care and Use Committee.

ELISA.

ELISA was conducted as previously reported (Hudalla 2013), with minimalmodifications. For mice immunized with βTail-GFP, serum collected atweek 7 was analyzed; for mice immunized with βTail-GFP, serum collectedat week 10 was analyzed. For all total IgG ELISAs, following overnightcoating with 1 μg/mL βTmutant-GFP in PBS or PBS (negative control), allplates were washed 3 times with 0.5% Tween-20 in PBS. Wells were thenblocked with 200 μL of 1% BSA/0.5% Tween-20 in PBS for 1 h at roomtemperature. This solution was removed from the wells, 100 μL of serumdiluted 1:10²-1:10⁹ in PBS with 1% BSA was then added directly to thewells without washing, and the plate was incubated for 1 h at roomtemperature. At the end of serum binding, the solution was removed fromthe plate, and the plates were washed 5 times with 0.5% Tween-20 in PBS.100 μL of peroxidase-conjugated goat anti-mouse IgG (H+L) (JacksonImmuno Research, cat 115035003) diluted 1:5000 in PBS with 1% BSA wasthen added to the wells, and the plates were incubated for 45 min atroom temperature. At the end of secondary antibody binding, the solutionwas removed and plates were washed 5 times with 0.5% Tween-20 in PBS.Plates were then developed by adding 100 μL of TMB substrate(eBioscience CA, cat 00-4201-56), incubating for 7.5 min at roomtemperature, then quenching the reaction with 50 μL of 1 M H₃PO₄.Absorbance was then measured at 450 nm with a SpectraMax M5 plate reader(Molecular Devices, CA).

For all isotyping ELISAs, following overnight coating with 1 μg/mLβTmutant-GFP in PBS or PBS, all plates were washed 3 times with 0.5%Tween-20 in PBS. Wells were then blocked with 200 μL of 1% BSA/0.5%Tween-20 in PBS for 1 h at room temperature. This solution was removedfrom the wells, 100 μL of serum collected at week 7 was diluted 1:500 inPBS with 1% BSA was then added directly to the wells without washing,and the plate was incubated for 1 h at room temperature. At the end ofserum binding, the solution was removed from the plate, and the plateswere washed 3 times with 0.5% Tween-20 in PBS. 100 μL of goat anti-mouseIgG1 (Sigma cat M5532), IgG2a/c (M5657), IgG2b (M5782), IgG3 (M5907), orIgM (M6157) diluted 1:1000 in PBS with 1% BSA was then added to thewells, and the plates were incubated for 30 min at room temperature. Atthe end of primary antibody binding, the solution was removed from theplate, and the plates were washed 3 times with 0.5% Tween-20 in PBS. 100μL of peroxidase-conjugated rabbit anti-goat IgG diluted 1:5000 in PBSwith 1% BSA was then added to the wells, and the plates were incubatedfor 15 min at room temperature. At the end of secondary antibodybinding, the solution was removed from the plate, and the plates werewashed 3 times with 0.5% Tween-20 in PBS. Plates were then developed byadding 100 μL of TMB substrate, incubating for 5 min at roomtemperature, then quenching the reaction with 50 μL of 1 M H₃PO₄.Absorbance was then measured at 450 nm with a SpectraMax M5 platereader.

Example 3 βTail Fusion Proteins

All recombinant fusion protein genes were inserted between the NcoI andXhoI sites in pET-21d. Genetic and amino acid sequences for each βTailfusion protein are provided below.

βTail-GFP GeneticSequence: [SEQ ID NO. 64]ATGGCCCTGAAAGTGGAACTGGAAAAACTGAAAAGCGAACTGGTGGTGCTGCATAGCGAACTGCATAAACTGAAAAGCGAACTGGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGCGGTGGAGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAACTCGAGCACCACCACCACCACCACTGA Amino Acid Sequence:[SEQ ID NO. 65]M A L K V E L E K L K S E L V V L H S E L H K L K S E L G S G GG G S G G G G S G G G G G G G S G G G G S G G G G S G G G G S SK G E E L F T G V V P I L V E L D G D V N G H K F S V S G E G EG D A T Y G K L T L K F I C T T G K L P V P W P T L V T T F S YG V Q C F S R Y P D H M K R H D F F K S A M P E G Y V Q E R T IS F K D D G N Y K T R A E V K F E G D T L V N R I E L K G I D FK E D G N I L G H K L E Y N Y N S H N V Y I T A D K Q K N G I KA N F K I R H N I E D G S V Q L A D H Y Q Q N T P I G D G P V LL P D N H Y L S T Q S A L S K D P N E K R D H M V L L E F V T AA G I T H G M D E L Y K L E H H H H H H Stop βTmutant-GFPGenetic Sequence: [SEQ ID NO. 66]ATGGCCGGAAAACCAGAAGGAGAAAAACCAAAATCAGAAGGAGGACCAGGACACTCAGAAGGACACAAACCAAAATCAGAAGGAGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGCGGTGGAGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAACTCGAGCACCACCACCACCACCACTGA Amino Acid Sequence:[SEQ ID NO. 67]M A G K P E G E K P K S E G G P G H S E G H K P K S E G G S G GG G S G G G G S G G G G G G G S G G G G S G G G G S G G G G S SK G E E L F T G V V P I L V E L D G D V N G H K F S V S G E G EG D A T Y G K L T L K F I C T T G K L P V P W P T L V T T F S YG V Q C F S R Y P D H M K R H D F F K S A M P E G Y V Q E R T IS F K D D G N Y K T R A E V K F E G D T L V N R I E L K G I D FK E D G N I L G H K L E Y N Y N S H N V Y I T A D K Q K N G I KA N F K I R H N I E D G S V Q L A D H Y Q Q N T P I G D G P V LL P D N H Y L S T Q S A L S K D P N E K R D H M V L L E F V T AA G I T H G M D E L Y K L E H H H H H H Stop βTail-dsREDGenetic Sequence: [SEQ ID NO. 68]ATGGCCGGAAAACCAGAAGGAGAAAAACCAAAATCAGAAGGAGGACCAGGACACTCAGAAGGACACAAACCAAAATCAGAAGGAGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGCGGTGGAGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTAGTGACGACAACACCGAGGACGTCATCAAGGAGTTCATGCAGTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCCACTACTTCGAGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGAGGGCACCCAGACCGCCAAGCTGCAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACATGAAGCTGTCCTTCCCCGAGGGCTTCACCTGGGAGCGCTCCATGAACTTCGAGGACGGCGGCGTGGTGGAGGTGCAGCAGGACTCCTCCCTGCAGGACGGCACCTTCATCTACAAGGTGAAGTTCAAGGGCGTGAACTTCCCCGCCGACGGCCCCGTAATGCAGAAGAAGACTGCCGGCTGGGAGCCCTCCACCGAGAAGCTGTACCCCCAGGACGGCGTGCTGAAGGGCGAGATCTCCCACGCCCTGAAGCTGAAGGACGGCGGCCACTACACCTGCGACTTCAAGACCGTGTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCAACCACTACGTGGACTCCAAGCTGGACATCACCAACCACAACGAGGACTACACCGTGGTGGAGCAGTACGAGCACGCCGAGGCCCGCCACTCCGGCTCCCAGCTCGAGCACCACCACCACCACCACTGA Amino Acid Sequence: [SEQ ID NO. 69]M A L K V E L E K L K S E L V V L H S H L E K L K S E L G S G GG G S G G G G S G G G G G G G S G G G G S G G G G S G G G G S SD D N T E D V I K E F M Q F K V R M E G S V N G H Y F E I E G EG E G K P Y E G T Q T A K L Q V T K G G P L P F A W D I L S P QF Q Y G S K A Y V K H P A D I P D Y M K L S F P E G F T W E R SM N F E D G G V V E V Q Q D S S L Q D G T F I Y K V K F K G V NF P A D G P V M Q K K T A G W E P S T E K L Y P Q D G V L K G EI S H A L K L K D G G H Y T C D F K T V Y K A K K P V Q L P G NH Y V D S K L D I T N H N E D Y T V V E Q Y E H A E A R H S G SQ H H H H H H Stop βTail-eGFP Genetic Sequence: [SEQ ID NO. 70]ATGGCACTGAAAGTCGAACTGGAAAAACTGAAATCGGAACTGGTCGTCCTGCACTCGCACCTGGAAAAACTGAAATCGGAACTGGGTAGCGGTGGCGGTGGCTCTGGTGGCGGTGGCAGTGGTGGCGGTGGCGGTGGCGGTTCCGGCGGTGGCGGTTCAGGCGGTGGCGGTTCGGGCGGTGGCGGTAGCTCTATGGTTAGCAAAGGTGAAGAACTGTTTACCGGCGTGGTTCCGATTCTGGTCGAACTGGATGGTGACGTGAATGGCCATAAATTCAGTGTGTCCGGCGAAGGTGAAGGCGATGCGACCTATGGTAAACTGACGCTGAAATTTATCTGCACCACGGGTAAACTGCCGGTTCCGTGGCCGACCCTGGTCACCACGCTGACGTATGGTGTCCAGTGTTTCAGCCGCTACCCGGATCATATGAAACAACACGACTTTTTCAAATCTGCGATGCCGGAAGGTTATGTGCAGGAACGTACCATTTTCTTTAAAGATGACGGCAACTACAAAACCCGCGCCGAAGTGAAATTTGAAGGTGATACGCTGGTTAACCGTATTGAACTGAAAGGCATCGATTTCAAAGAAGACGGTAATATCCTGGGCCATAAACTGGAATACAACTACAACTCACACAACGTCTACATTATGGCAGATAAACAGAAAAACGGTATCAAAGTGAACTTCAAAATCCGCCATAATATCGAAGATGGCTCCGTTCAACTGGCTGACCACTATCAGCAAAACACCCCGATTGGTGATGGCCCGGTTCTGCTGCCGGACAATCATTACCTGTCAACGCAGTCGGCACTGAGCAAAGATCCGAACGAAAAACGTGACCACATGGTTCTGCTGGAATTTGTCACGGCTGCGGGTATTACGCTGGGTATGGACGAACTGTATAAACTCGAG Amino Acid Sequence: [SEQ ID NO. 71]M A L K V E L E K L K S E L V V L H S H L E K L K S E L G S G GG G S G G G G S G G G G G G G S G G G G S G G G G S G G G G S SM V S K G E E L F T G V V P I L V E L D G D V N G H K F S V S GE G E G D A T Y G K L T L K F I C T T G K L P V P W P T L V T TL T Y G V Q C F S R Y P D H M K Q H D F F K S A M P E G Y V Q ER T I F F K D D G N Y K T R A E V K F E G D T L V N R I E L K GI D F K E D G N I L G H K L E Y N Y N S H N V Y I M A D K Q K NG I K V N F K I R H N I E D G S V Q L A D H Y Q Q N T P I G D GP V L L P D N H Y L S T Q S A L S K D P N E K R D H M V L L E FV T A A G I T L G M D E L Y K H H H H H H Stop βTail-cutinaseGenetic Sequence: [SEQ ID NO. 72]ATGGCCCTGAAAGTGGAACTGGAAAAACTGAAAAGCGAACTGGTGGTGCTGCATAGCGAACTGCATAAACTGAAAAGCGAACTGGGATCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTAGATCTGGCCTGCCTACTTCTAACCCTGCCCAGGAGCTTGAGGCGCGCCAGCTTGGTAGAACAACTCGCGACGATCTGATCAACGGCAATAGCGCTTCCTGCGCCGATGTCATCTTCATTTATGCCCGAGGTTCAACAGAGACGGGCAACTTGGGTACCCTCGGTCCTAGCATTGCCTCCAACCTTGAGTCCGCGTTCGGCAAGGACGGTGTCTGGATTCAGGGCGTTGGCGGTGCCTACCGTGCCACTCTTGGAGACAATGCTCTCCCTCGCGGAACCTCTAGCGCCGCAATCAGGGAGATGCTCGGTCTCTTCCAGCAGGCCAACACCAAGTGCCCTGACGCGACTTTGATCGCCGGTGGCTACAGCCAGGGTGCTGCACTTGCAGCCGCCTCCATCGAGGACCTCGACTCGGCCATTCGTGACAAGATCGCCGGAACTGTTCTGTTCGGCTATACCAAGAACCTACAGAACCGTGGCCGAATCCCCAACTACCCTGCCGATAGGACCAAGGTCTTCTGCAATACAGGGGATCTCGTTTGTACTGGTAGCTTGATCGTTGCTGCACCTCACTTGGCGTATGGTCCTGATGCTCGTGGCCCTGCCCCTGAGTTCCTCATCGAGAAGGTTCGGGCTGTCCGTGGTTCTGCTCTCGAGCACCACCACCACCACCACTGAAmino Acid Sequence: [SEQ ID NO. 73]M A L K V E L E K L K S E L V V L H S E L H K L K S E L G S G GG G S G G G G S G G G G S R S G L P T S N P A Q E L E A R Q L GR T T R D D L I N G N S A S C A D V I F I Y A R G S T E T G N LG T L G P S I A S N L E S A F G K D G V W I Q G V G G A Y R A TL G D N A L P R G T S S A A I R E M L G L F Q Q A N T K C P D AT L I A G G Y S Q G A A L A A A S I E D L D S A I R D K I A G TV L F G Y T K N L Q N R G R I P N Y P A D R T K V F C N T G D LV C T G S L I V A A P H L A Y G P D A R G P A P E F L I E K V RA V R G S A L E H H H H H H Stop

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-60. (canceled)
 61. A method of inducing an immune response comprisingadministering an effective amount of a composition, wherein thecomposition comprises a β-sheet nanofiber complex composition comprisinga β-sheet nanofiber structure comprising: a) a plurality of non-β-sheetpeptide tags that undergo a transition from a non-β-sheet structure to aβ-sheet structure in the presence of β-sheet peptides, wherein thenon-β-sheet peptide tags comprise an amino acid sequence having at least90% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1),LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3),LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL(SEQ ID NO:5), wherein at least one of the non-β-sheet peptide tags isattached to an antigen; and b) a plurality of β-sheet peptides. 62-63.(canceled)
 64. A method of culturing a cell, comprising incubating thecell in a cell culture medium comprising a β-sheet nanofiber complexcomposition comprising a β-sheet nanofiber structure comprising: a) aplurality of non-β-sheet peptide tags that undergo a transition from anon-β-sheet structure to a β-sheet structure in the presence of β-sheetpeptides, wherein the non-β-sheet peptide tags comprise an amino acidsequence having at least 90% identity with the sequence ofLVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2),LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL(SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5), wherein atleast one of the non-β-sheet peptide tags is attached to an antigen; andb) a plurality of β-sheet peptides.
 65. The method of claim 61, whereinthe non-β-sheet peptide tag comprises an amino acid sequence having atleast 95% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1),LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3),LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL(SEQ ID NO:5).
 66. The method of claim 61, wherein the non-β-sheetpeptide tag comprises the amino acid sequence of LVVLHSELHKLKSEL (SEQ IDNO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ IDNO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), orLKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5).
 67. The method of claim 61,wherein the non-β-sheet peptide tag consists of the amino acid sequenceof LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2),LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL(SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5).
 68. Themethod of claim 61, wherein the non-β-sheet peptide tag is 14 to 56amino acids in length.
 69. The method of claim 61, wherein thenon-β-sheet peptide tags and β-sheet peptides have a molar ratio ofabout 1:10 to about 1:10,000.
 70. The method of claim 61, wherein one ormore of the β-sheet peptides are 2 to 40 amino acids in length.
 71. Themethod of claim 61, wherein one or more of the β-sheet peptides comprisean amino acid sequence having at least 90% identity with the sequence ofQQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ IDNO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ(SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ IDNO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14);FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16);AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18);AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20);RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23);WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK(Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO.26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV(SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF;Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQID NO. 33).
 72. The method of claim 61, wherein one or more of theβ-sheet peptides comprise an amino acid sequence having at least 95%identity with the sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ(SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9);QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11);FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13);QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE(SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ IDNO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO.19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21);SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (WhereX=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P)(SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO.25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO.27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV(SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO.32); or YTIAALLSPY (SEQ ID NO. 33).
 73. The method of claim 61, whereinone or more of the β-sheet peptides comprise the amino acid sequence ofQQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ IDNO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ(SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ IDNO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14);FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16);AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18);AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20);RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23);WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK(Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO.26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV(SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF;Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQID NO. 33).
 74. The method of claim 61, wherein one or more of theβ-sheet peptides consist of the amino acid sequence of QQKFQFQFEQQ (SEQID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE(SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO.11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13);QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE(SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ IDNO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO.19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21);SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (WhereX=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P)(SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO.25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO.27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV(SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO.32); or YTIAALLSPY (SEQ ID NO. 33).
 75. The method of claim 61, whereinone or more of the β-sheet peptides comprise a plurality ofself-assembling peptides.
 76. The method of claim 61, wherein theantigen is an enzyme, fluorescent protein, cell binding domain, celladhesion domain, extracellular matrix domain, reporter protein,cytokine, antigen, signaling domain, immunomodulating protein,cross-linking protein, hormone, hapten, or a combination thereof. 77.The method of claim 61, wherein at least one of the non-β-sheet peptidetags attached to an antigen is further defined as a fusion protein. 78.The method of claim 61, wherein one or more of the non-β-sheet peptidetag are attached to the amino-terminus of an antigen peptide.
 79. Themethod of claim 61, wherein the β-sheet nanofiber complex compositionhas a length of at least about 0.2 to about 100 μm.
 80. The method ofclaim 61, wherein the β-sheet nanofiber complex composition has amolecular weight of at least about 10,000 da to about 7×10⁸ da.
 81. Amethod of preparing a nanofiber complex composition, comprising mixingthe following: a) a plurality of non-β-sheet peptide tags, wherein thenon-β-sheet peptide tags comprise an amino acid sequence having at least90% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1),LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3),LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL(SEQ ID NO:5), wherein a non-β-sheet peptide tag is attached to anantigen, and b) a plurality of β-sheet peptides, under conditions thatallow the non-β-sheet peptide tags to transition from a non-β-sheetstructure to a β-sheet structure, thereby preparing a nanofiber complexcomposition that forms a β-sheet structure comprising the non-β-sheetpeptide tags and β-sheet peptides.
 82. The method of claim 81, furthercomprising shaking the mixture, thereby forming a microgel.